Gene cloning

Cloning means asexual reproduction to obtain organism that are genetically

identical to one another and to the parent.

Of course this contrast with sexual reproduction where the offspring are not
usually identical

Clones are genetically identical but their appearance may change other factors
of environment.

These points applicable to bacteria to human

The plants propagated through cuttings are clones

Similarly picking a single colony from a bacterial cultures and multiplying the
same is cloning

The term cloning also applies to genes.

Introducing a gene in to bacterial cell and multiplying the number of copies of

the gene by replicating or growing the bacterial cell culture is gene cloning. The
multiplied gene copies can be isolated from the cell and purified genes can be
used for other purposes.
Procedures of gene cloning

Some bacterial species will naturally take up DNA by process known as

transformation

Some species needs physical or chemical treatment to take up DNA

DNA taken into the cells must integrate in to genome otherwise it will not be
multiplied.

A small unit of extra chromosomal DNA called plasmids can integrate this
DNA and send inside the cell and multiply the DNA inserted in to plasmid.

This DNA material / plasmids are called vectors or cloning vehicle.

First the Vector DNA is cut or opened with restriction enzymes

The piece of DNA or the gene of interst is inserted in to vector with the help of
another enzyme called ligase

This ligated insert and vector DNA is called recombinant DNA

This recombinant DNA is send inside the bacterial cell called transformation.
The inserted bacterial cell is grown in culture.

After 30 generations (after 10 hours) it will be multiplied in to 10 9 cells (100

crores of cells) is produced thus 100 crores of DNA copies were created.

Hence DNA modification enzymes are very important for this work.

Restriction endonucleases (RE)

RE, the enzymes that are used to cut DNA molecules in specific places

Phage grown in E.coli strain C will not infect the E.coli strain K, Ecoli-K
restricts the growth of this phage

E.coli-K produces a enzyme called restriction endonuclease that cut and destroy
the phage DNA and stop the growth of the phage

Restriction - Because for the way they work, they restrict virus to only
one host bacterial strain. They are also restricted to acting on only
specific DNA sequences

Endonuclease - They cut nucleic acids in the middle not just the ends
 Restriction enzymes are endonucleases that cut double-stranded
DNA

Restriction systems allow bacteria to monitor the origin of incoming

DNA and to destroy it if it is recognized as foreign

Specificity

Restriction endonucleases recognize specific sequences in the incoming

DNA and cleave the DNA into fragments

Types of Restriction enzymes

Type I

One enzyme with different subunits for recognition, cleavage and

methylation. Recognizes and methylates a single sequence but cleaves
DNA up to 1000 bp away

The active enzyme consists of two restriction subunits, two modification

(methylation) subunits and one recognition subunit.

Because of the methylation reaction is performed by the same enzyme

which mediates cleavage, the target DNA may be modified before it is
cut.

These features mean that type I systems are of little value for gene
manipulation
Type-II Restriction enzyme

Two different enzymes which both recognize the same target sequence,
which is symmetrical. The two enzymes either cleave or modify the
recognition sequence

Type II restriction enzymes cut the DNA in the middle of the recognition
site

Because of their specificity and exact position they are used in genetic
engineering experiments

There are two different ways of cutting the recognition site in half.

One way is to cut both strands of the double stranded DNA at the same
point.

The alternative is to cut the two strands in different places, which

generates overhanging ends

Type III

One enzyme with two different subunits, one for recognition and
modification and one for cleavage. Recognizes and methylates same
sequence but cleaves 2426 bp away

Type III enzymes have symmetrical recognition sequences but otherwise

resemble type I systems and are of little
value

Two different ways of cutting If two different pieces of

DNA are cut with the same

restriction enzyme or
enzymes that generate the
same

overhang, the same sticky

ends are generated

Advantage of Type II
 restriction and modification are mediated by separate enzymes so it is
possible to cleave DNA in the absence of modification

The restriction activities do not require cofactors such as ATP or S-

adenosylmethionine, making them easier to use.

Type II enzymes recognize a defined, usually symmetrical, sequence and

cut within it.

Many of them also make a staggered break in the DNA

Nomenclature

The species name of the host organism is identified by the first letter of
the genus name

The first two letters of the specific epithet to generate a three letter
abbreviation

This abbreviation is always written in italics.

When a particular host strain has several different R-M systems, these are
identified by roman numerals
Recognition sequences

Most, type II restriction endonucleases recognize and cleave DNA within

particular sequences of four to eight nucleotides which have a twofold
axis of rotational symmetry.

Such sequences are often referred to as palindromes because of their

similarity to words that read the same backwards as forwards
Variations on cutting and joining DNA molecules

In order to join two fragments of DNA together, it is not essential that

they are produced by the same restriction endonuclease.

Many different restriction endonucleases produce compatible cohesive

ends.

For example, AgeI (A/CCGGT) and AvaI C/CCGGG) produce molecules with
identical 5 overhangs and so can be ligated together

Isoschizomers

An additional element of flexibility arises from the existence of several

enzymes that recognizes the same sequence of bases are known as
isoschizomers.

Greek Iso- equal, skizho- split

In many cases isoschizomers not only have the same recog site but cut in the
same place within the recognition sequence.

For example Acc65I and KpnI both recognize the sequence GGTACC but cut at
different place

But xmaI and sma I

Modifying enzymes

DNA polymerase enzyme is responsible for constructing new DNA

strands.

DNA polymerase enzyme catalyses the addition or synthesis of new

complementary strands using a template strand of DNA and all four
deoxyribonucleotide triphosphate.

There are three major DNA polymerase enzymes that will polymerize
nucleotides into a growing strand of DNA in E.coli.

These enzymes are DNA polymerase I, II and III.

DNA pol I and II are primarily used for DNA repair and for replicating a
small length or segment of DNA.

DNA polymerase that have been studied have two basic

requirements:

1. A template DNA strand to copy.

2. A primer strand to which nucleotides can be added.

Likewise all DNA polymerase enzymes have two basic properties:

1. 5' -3' polymerization capacity

2. 5' -3' and exonuclease activity

 The 5'-3' exonuclease activity of DNA pol I differs from the 3' -5'
exonuclease used by both DNA pol I and pol III in two
different/important ways.

Firstly 5'-3' exonuclease can remove one nucleotide at a time from a

region of DNA that is improperly base-paired. The nucleotides it removes
can be either ribonucleotides or deoxyribonucleotide.

Secondly, 5' -3 ' exonuclease can also remove groups of altered

nucleotides in 5 ' -3 ' direction, removing from one to ten nucleotides at a
time. This ability is important in the repair of damaged DNA.

A typical bacterial cell contains approximately 300 to 400 molecules of

DNA pol I per cell and only about 40 copies of DNA polymerase II and
10 copies of DNA pol III.

Eukaryotes have 15 types of DNA

polymerases

Klenow Fragment of E. coli DNA

Polymerase I

The 5' -> 3' exonuclease activity of E.

coli's DNA Polymerase I makes it
unsuitable for many applications.

Exposure of DNA polymerase I to the

protease subtilisin cleaves the molecule
into a small fragment, which retains the 5'
-> 3' exonuclease activity, and a large
piece called Klenow fragment.

The large or Klenow fragment of DNA polymerase I has DNA

polymerase and 3' -> 5' exonuclease activities, and is widely used in
molecular biology.
Functions

Synthesis of double-stranded DNA from single-stranded templates

it can displace primers downstream and continue synthesizing new DNA.

A "fill-in" reaction is used to create blunt ends on fragments created by

cleavage with restriction enzymes that leave 5' overhangs.

Digesting away protruding 3' overhangs: yielding blunt ends

DNA Polymerase II

DNA polymerase II (also known as DNA Pol II or Pol II) is

a prokaryotic DNA Polymerase most likely involved in DNA repair.

Pol II helps to overcome the problem because it can reinitiate DNA

synthesis downstream of gaps.

Pol II has a low error rate but it is much too slow to be of any use in
normal DNA synthesis.

Pol II differs from Pol I in that it lacks a 5'->3' exonuclease activity

DNA Polymerase III

DNA polymerase III holoenzyme is the primary enzyme complex

involved in prokaryotic DNA replication.

Being the primary holoenzyme (with all subunits and cofactors attached)
involved in replication activity, the DNA Pol III holoenzyme also has
proofreading capabilities that correct replication mistakes by means of
exonuclease activity working 3'->5'.

DNA Pol III is a component of the replisome, which is located at the

replication fork.

T4 DNA Polymerase

The activities of T4 DNA polymerase are very similar to Klenow

fragment of DNA polymerase I - it functions as a 5' -> 3' DNA
polymerase and a 3' -> 5' exonuclease, but does not have 5' -> 3'
exonuclease activity.

T4 DNA polymerase is used for blunting the ends of DNA with 5' or 3'
overhangs

The 3' -> 5' exonuclease activity of T4 DNA polymerase is roughly 200
times that of Klenow fragment

DNA polymerase will not displace downstream oligonucleotides like

klenow fragments
T7 DNA Polymerase

The DNA polymerase of T7 bacteriophage has DNA polymerase and 3' -

> 5' exonuclease activities, but lacks a 5' -> 3' exonuclease domain.

Mostly similar to Klenow and T4 DNA polymerase

The claim to fame for T7 DNA polymerase is it's processivity.

DNA synthesized before the enzyme dissociates from the template is

considerably greater than for other enzymes Used in DNA sequencing

Terminal Transferase

Terminal transferase catalyzes the addition of nucleotides to the 3'

terminus of DNA

It works on single-stranded DNA, including 3' overhangs of double-

stranded DNA

Does not require a primer.

It can also add homopolymers of ribonucleotides to the 3' end of DNA

Thermostable DNA Polymerases

Taq polymerase purified from the hot springs bacterium Thermus

aquaticus, was published in 1976.

Roughly 10 years later, the polymerase chain reaction was developed and
shortly thereafter "Taq" became a household word in molecular biology
circles.
 Currently, the world market for Taq polymerase is in the hundreds of
millions of dollars each year.

The thermophilic DNA polymerases, like other DNA polymerases,

catalyze template-directed synthesis of DNA from nucleotide
triphosphates.

It lacks a 3 to 5 exonuclease proofreading activity

A primer having a free 3' hydroxyl is required to initiate synthesis and
magnesium ion is necessary.
In general, they have maximal catalytic activity at 75 to 80C, and
substantially reduced activites at lower temperatures.
One of the most discussed characteristics of Taq polymerases is their
error rate.
polymerases lacking 3'->5' exonuclease activity generally have higher
error rates than the polymerases with exonuclease activity.
The total error rate of Taq polymerase has been variously reported
between 1 x 10-4 to 2 x 10-5 errors per base pair.
Pfu polymerase appears to have the lowest error rate at roughly 1.5 x 10-
6
error per base pair
Types of Taq Polymerase

Polymerase 3'->5' Source and Properties

Exonuclease

Taq No From Thermus

aquaticus. Halflife at
95C is 1.6 hours.

Pfu Yes From Pyrococcus

furiosus. Appears to have
the lowest error rate of
known thermophilic
DNA polymerases
Vent Yes From Thermococcus
litoralis; also known as
Tli polymerase. Halflife
at 95 C is approximately
7 hours.
Reverse transcriptase

Reverse transcriptase, also known as RNA-dependent DNA

polymerase, is a DNA polymerase enzyme that transcribes single-
stranded RNA into double-stranded DNA.

First RNA has been reverse transcribed into a single strand cDNA then
second strand has been created with the help of DNA polymerase

Reverse transcriptases have two activities:

DNA polymerase activity: it will transcribe both single-stranded RNA

RNase H activity: RNase H is a ribonuclease that degrades the RNA from

RNA-DNA hybrids, such as are formed during reverse transcription of an
RNA template. This enzyme functions as both an endonuclease and
exonuclease in hydrolyzing its target

Another common use for reverse transcriptase is to generate DNA copies

of RNAs prior to amplifying that DNA by polymerase chain reaction
(PCR). Reverse Transcription PCR, usually called simply RTPCR

Types of Reverse transcriptase

Moloney murine leukemia virus: a single polypeptide

Avian myeloblastosis virus: composed of two peptide chains

Nucleases: DNase and RNase

A nuclease is an enzyme capable of cleaving the phosphodiester

bonds between the nucleotide subunits of nucleic acids
 Dnase : Cleaves and digests DNA molecules

RNase : Cleaves and digests RNA molecules

Numerous types of DNase and RNase have been isolated and

characterized.

They differ among other things in substrate specificity, cofactor

requirements, and whether they cleave nucleic acids internally
(endonucleases), chew in from the ends (exonucleases) or attack in both
of these modes.

DNase

The most widely used nucleases are DNase I and RNase A, both of
which are purified from bovine pancreas

Deoxyribonuclease I cleaves double-stranded or single stranded DNA.

Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues,
and the enzyme is therefore an endonuclease. Major products are 5'-
phosphorylated di, tri and tetranucleotides.

Some of the common applications of DNase I are:

Eliminating DNA (e.g. plasmid) from preparations of RNA.

Analyzing DNA-protein interactions via DNase footprinting.

Nicking DNA prior to radiolabelling by nick translation.

RNase

Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA

at the 3' end of pyrimidine residues. It degrades the RNA into 3'-
phosphorylated mononucleotides and oligonucleotides.

Some of the major use of RNase A are:

Eliminating or reducing RNA contamination in preparations of plasmid

DNA.

Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave

the RNA in RNA:DNA hybrids at sites of single base mismatches, and the
cleavage products can be analyzed.
Ligation Strategy-Joining DNA molecules

DNA fragments can be joined to create artificially recombinant molecules.

The joining or ligation of DNA molecules is carried out by an enzyme

known as DNA ligase

The natural role of DNA ligase is to repair single strand breaks (nicks)

and joining the lagging strand during DNA replication.

The action of ligase requires that the nick should expose 3'-OH group and a
5'-phosphate group

Digestion with restriction enconuclease cuts the DNA in this way -i.e it
leaves the phosphate on the 5' position of the deoxyribose.

T4 DNA Ligase (encoded by bacteriophage T4) capable of ligating blunt ended

DNA fragments

This is the Ligase which is used most

extensively

T4 Ligase requires ATP as a co substrate

in the first step, the ligase reacts with ATP and

forms enzyme -AMP comlex to the phosphate
group

Then it attack the 3'OH group, forming a new

covalent phospodiesterbond and released AMP

Ecoli. DNA Ligase

E. coli enzyme requires NAD+ Cofactor

It ligates only the overhangs or cohesive fragments and nicks.

Optimizing ligation condition

Ligation is the most un predictable step

The contaminations in DNA preparations and quality and degradation of the

enzyme affects he ligation
Most important factor is the reaction temperature. Earlier works recommended
10C which needs long incubation

The latest protocols recommends 16 C

The vector and insert conscentration is very important -Lower concentration

slowdown the reaction.

Equal molar concentration is desirable

For example if a vector is 5kb and insert is 500 base then 1:1 molar is 500ng
vector and 50ng insert.

Providing more insert will increase the possibility of obtaining multipple insert.

Use of Alkaline Phosphotase

Ligation process depends absolutely on the presence of 5' phosphate at the nick
site.

Alkaline phosphotase (Calf intestinal phosphotase-CIP) removes the 5'

phosphate.

Treating the vector with this enzyme is essential thus phophate is removed and
the insert with phosphate molecule can ued for ligation

But this treatment stops the sealing in other fragment of DNA but it is not a
problem - after transfromation the cell system seals the nick.

Modification of Restriction fragment ends

Restriction fragments with sticky ends are useful as they can be readily ligated.

however , the limitation is tey ligate only with compatible ends

So EcoRI fragment can be ligated with another EcoRI fragment but not BamHI.

In such a condition following methods are empoyed

1. Trimming and filling

Convert the sticky ends to blunt end

DNA plymerase fills the gap in the sticky end fragments and make them to
blunt end
E.Coli DNA polymeraseI have exonuclease activity but klwnow does not have
exonuclease activity so the klwnow fragment could be used for filling which
would make the overhang in to blunt end.

To remove overhanng T4 DNA Polymerase could be used to remove overhang.

Linkers

Still some way short of the complete versatality

that is our goal especially blunt end ligation is so
inefficient, and requires much concentration of
DNA.

The linking process can be made much more

efficient and versatile by the use of linkers

These are short piece of DNA (8-14bp) that

contain a restriction site

For example CCGGATCCGG contains the

BamHI site (GGATCC)

further ist is self complementary single strand so

that we can produce double stranded DNA 10
base pairs long
This strand is joined to our insert molecule by blunt end ligation and we have
the extra BamHI site near each end (the high molecular concentration of linkers
encourage this reaction)

Cutting this with BamHI will generate a sticky end and canbe ligated with the
vector digested with Bam HI digested.

Further this technology have the versatality and we can use any type of linkers
with different restriction site.

Adapters

Alternatively an adapter with one sticky end

and one blunt end can be used to convert blunt
ended DNA fragments

Adaptors are short double strand DNA

molecules which are predigested by the
appropriate enzymes

It attaches to Vector DNA and produces

overhang ends

Homopolymer tailing

A general method for joining DNA molecules makes use of the annealing of
complementary homopolymer sequences.

Thus, by adding oligo(dA) sequences to the3 ends of one population of DNA

molecules and oligo(dT) blocks to the 3 ends of another population, the two
types of molecule can anneal to form mixed dimeric circles

An enzyme purified from calf thymus, terminal deoxynucleotidyltransferase,

provides the means by which the homopolymeric extensions can be synthesized,
for if presented with a single deoxynucleotide triphosphate it will repeatedly
add nucleotides to the 3 OH termini of a population of DNA molecules
Joining of PCR Product (TA cloning)

Some of the Polymerases used in PCR amplification also have terminal

transferase action, they add a single adenosine residue to the 3 ends

Commercially available TA vectors which are supplied in linearized form with

a single 3-T overhandg which is compatible with 3A PCR product
A diagram that shows the location of restriction enzyme cut sites on a segment
of DNA is known as a restriction map.

The first step in generating such a map is to digest the DNA with a series of
restriction enzymes, one at a time.

The fragments of digested DNA are separated by agarose gel electrophoresis

Comparison with appropriate standards allows the sizes of the fragments to be

estimated. This reveals how many recognition sites each enzyme has in the
DNA and their distances apart

What remains unknown is the relative order of the fragments.

Unit II CLONING VECTORS

Vectors

A cloning vector is a small piece of DNA into which a foreign DNA fragment
can be inserted

Desirable properties of plasmid cloning vectors

It should be low molecular weight, so that it is easier to handle, isolate, no

shearing, If the vector is so big, it may have the chances of Restriction site
inside the vector which is not a desirable character.

It should be maintained in multipple copies in the host cell.

Should have the ability to confer readily selectable phenotypic traits on host
cells

Single sites for a large number of

restriction endonucleases Multipple
cloning sites

It should have the spectial charater to

identify the recombinant cells- like
Alpha complementation- LacZ system

It should have the antibiotic selection

genes in order to avaoid non
recombinants ( kanamycin, tetracycline,
geneticin, Ampicilin etc.)

Alpha complementation

The concept of alpha-

complementation is important because
it is a quick, easy, 1-step process of
determining whether a transformed
bacterial colony has plasmid+insert or
not (The tetracycline resistance of
pBR322 works, it is just time
consuming).

The key to alpha-complementation is the fact that the lac-Z gene product (B-
galactosidase) is a tetramer, and each monomer is made of two parts - lacZ-
alpha, and lacZ-omega. Researchers determined that if the alpha fragment was
deleted, the omega fragment is non-functional; however, alpha fragment
functionality can be restored in-trans via plasmid. Hence, then name alpha-
complementation.

What is needed for this to work as a cloning technique is a strain of E. colithat

has the deletion of the lac Z-alpha (lacZ DM15 works well as a genotype), and a
plasmid with the lacZ-alpha fragment as the scorable marker (such as
pBluescript or pCR2.1). If plain plasmid is successfully transformed into a cell,
then the cell will express functional B-galactosidase. However, if the
plasmid+insert is transformed into a cell, then it will express non-functional B-
galactosidase (the lac Z-alpha will be disrupted with the insert gene product).
Plate the cells out onto selection media based on the selectable marker, IPTG
(induces lac repressor to disengage), and X-gal (chromogenic substrate that
yields blue product when cleaved by B-galactosidase) and the white colonies
(non-functional B-galactosidase) are the ones with plasmid + insert; the blue
ones have plain plasmid.

Plasmid Vectors

Plasmid vectors are most used, versatile, easily manipulated vector

They are the work horses of Molecular biology

Plasmids are replicons which are stably inherited in an extra chromosomal state

Most plasmids exist as double-stranded circular DNA molecules

Plasmids occur widely in nature and are found in most bacterial species. They
are circular supercoiled double stranded structure.

If both strands of DNA are intact circles the molecules are described as
covalently closed circles or CCC DNA

If only one strand is intact, then the molecules are described as open circles or
OC DNA.

When isolated from cells, covalently closed circles often have a deficiency of
turns in the double helix, such that they have a super coiled configuration

Not all plasmids exist as circular molecules. Linear plasmids have been found in
a variety of bacteria, e.g. Streptomyces sp

To prevent nuclease digestion, the ends of linear plasmids need to be protected

They vary in size from few thousand base pairs to hundred kilo base pairs ,
typically 2-5kb. Most notorious property of plasmid is antibiotic resistance

Plasmids can be categorized into one of two major type conjugative or non-
conjugative depending upon whether or not they carry a set of transfer genes,
called the tra genes, which promote bacterial conjugation.

Plasmids can also be categorized on the basis of their being maintained as

multiple copies per cell (relaxed plasmids) or as a limited number of copies per
cell (stringent plasmids).

Plasmids encode only a few of the proteins required for their own replication
and in many cases encode only one of them. All the other proteins required for
replication, e.g. DNA polymerases, DNA ligase, helicases, etc., are provided by
the host cell.

Those replication proteins that are plasmid-encoded are located very close to the
ori (origin of replication) sequences at which they act.

The host range of a plasmid is determined by its ori region. Plasmids whose ori
region is derived from plasmid Col E1 have a restricted host range: they only
replicate in enteric bacteria, such as E. coli, Salmonella, etc. Other promiscuous
plasmids have a broad host range and these include RP4 and RSF1010.
Plasmids of the RP4 type will replicate in most Gram-negative bacteria,

The copy number of a plasmid is determined by regulating the initiation of

plasmid replication. Close to the ori region there is a gene, called repA in
pSC101, which encodes the RepA protein. This protein, which is the only
plasmid encoded protein required for replication, binds to the iterons and
initiates DNA synthesis

The loss of plasmids due to defective partitioning is called segregative

instability.
plasmids are stably maintained because they contain a partitioning function,
par, which ensures that they are stably maintained at each cell division. Such
par regions are essential for stability of lowcopy- number plasmids

Plasmid incompatibility is the inability of two different plasmids to coexist in

the same cell in the absence of selection pressure. The term incompatibility can
only be used when it is certain that entry of the second plasmid
Over 30 incompatibility groups have been defined in E. coli and 13 for plasmids
of S. aureus.

Desirable properties of plasmid cloning vehicles

low molecular weight;

ability to confer readily selectable phenotypic traits
on host cells;
single sites for a large number of restriction
endonucleases, preferably in genes with a readily
scorable phenotype.

The advantages of a low molecular weight are several. First, the plasmid is
much easier to handle, i.e. it is more resistant to damage by shearing, and is
readily isolated from host cells. Secondly, lowmolecular- weight plasmids are
usually present as multiple copies

pBR322, a purpose-built cloning vehicle

Most commonly used plasmid cloning vector

-Created in 1977 by Bolivar and Rodriguez
(mexicans)
-pBR322 is 4361 base pairs in length
-contains a replicon region pMB1
-Ampicillin and tetracycline resistance
-unique restriction sites for more than forty
restriction enzymes.
-11 of these 40 sites lie within the tetR gene.
2 sites within the promoter of the tetR gene.
6 key restriction sites inside the ampR gene.

pUC series vectors

pUC19 is a plasmid cloning

vector created by Messing and co-
workers in the University of California.
p in the name stands for plasmid and UC represents the University in
which it was created.
It is a circular double stranded DNA and has 2686 base pairs.
Recombinants can be identified by Blue-white screening method (lacZ
gene)
Multiple cloning sites available in lacZ region
Phage vector

Bacteriophages are viruses that infect bacteria

Phages are constructed from two basic components

protein form a coat or capsid within which nucleic acid genome is enclosed and
a tail structure.

Two types of phages are used as vectors for cloning works

1. double stranded DNA phages- Lambda-phage (T2,T4,T6)

2. Single strand DNA phage- M13 Phage (174)

They contain origin of replication but lack machinery to produce protein.

They inject their DNA into bacterial cell, multiply their DNA, phage proteins
are synthesized and released the infectious phage particle by lysis.

Bactriophage

Lambda phage is capable of infecting E.Coli strain K-12.

A phage particle has a head-and-tail structure, consisting of a core of

DNA within a protein coat (capsid) that is joined to a helical protein
structure (tail).
 Phage is a temperate virus, in that its life cycle consists of two
pathways - lytic and lysogenic.

Cell lysis leads to release of 100 infective phage particles

DNA exist as linear double strand DNA molecule about 48.5kb long.

At each 5' end single stranded overhang at 12 bases length and these are
referred as cos sites.

They can able to form circular structure in the host cell and follows the
bidirectional replication

A roling circle mechanism produces multiple -length linear forms of double

stranded DNA. This is called as concatamers with regularly spaced cos sites.

Packing of newly replicated genomic dna into phage head particles is with the
help of cos site cleavage

According to the protein signals the lytic and lysogenic pathway is decided

IF a transcriptional repressor Protein encoded by CI gene inhibits this

transcription then lysogeny is favoured.

the process of bacteriophage introduced into E.coli cells is called transfection

and making plaques in the lawn of bacteria.

IF the phage genome that encourages the lysogenic growth is removed from
their DNA they became lytic form and produces clear plaques

If the lysogenic genes are not disturbed they procuce cloudy plaque.

Based on these morphological characters the insert is cloned into the genomic
DNA of phage and recombinants were selected.

In labda gt10 and gt11 has the Cl gene which decides the lysogenic pathway

where as in EMBL3 and EMBL4 phages the genes are red and gam genes

Steps in phage cloning

First the recipient vector is cleave with RE in the place of either Cl gene or red
and gam genes
about 25 Kb DNA can be ligated (inserted) in the place of these genes and
insertional inactivation takes place and the lysogenic character of this phage
inactivated

Hence the clones with inserted fragments produces the clear plaques and the
clone without recombinants produces the cloud plaque

After insertion of the foreign DNa the DNA materials are combined with viral
Head, tail and assembly proteins makes the new virus particles - this process is
called invitro packaging and this phage particle is ready to infect.

Some of the phage vectors are provided with gal+ (Blue white screening
system) the colourless plaques are having the insert.

Genetic map of bacteriophage

M13 Bacterioptiage

widely employed for generating

ssDNA for dideoxy DNA
sequencing
-single-stranded circular DNA (+)
Following infection of the E. coli host cell, the (+) strand serves as
template for the synthesis of the complementary (-) strand.
 The double stranded viral DNA in the E. coli host cell, referred to as the
replicative form (RF), is replicated and amplified to 100-200 copies per
cell.
In a later stage, the (+) strand continues to be synthesized, and the (-)
strand is prevented from replication.
The accumulated (+) strands are packaged with the viral proteins to
generate phage particles
On a bacterial lawn, the growth of M13 does not give clear plagues.
Instead, the plagues appear turbid, because Ml 3 is non-lytic
Cosmids
Cosmids were developed to overcome the technical problem of introducing
large piece of DNA in to E.Coli ( Larger plasmid can not be transferred to
E.coli through transformation).

Cosmids are plasmid vectors that contains bacteriophage Lambda cos sites.

These cos sites are responsible for the assembly of virus particle in to full virus
during lytic cyle.

The cosmid contains cos sites and a restriction site which help them to linearize.

They have usual antibiotic selection marker and bacterial origin of replications

The source DNA or the insert is partially digested to create large piece of DNA
around 40 Kb

The cosmid is digested with another enzyme ( in between two cos sites) and the
large piece of DNA is ligated.

This large ligated malecules with cos sites are recognized during packaging of
phage paticles ( the cos sites with more than 45 Kb is well recognized during
viral packaging)

The invitro packaging is carried out with head, tail, assembly proteins and this
ligated DNA molecules- during in vitro packaging the cos sites of this ligated
molecules are recognized and the recombinant plasmid is packaged in to the
bacteriophage virus.

This virus assembly is used to infect E.coli cells and the cosmids are being
transferred to E.coli cell through transfection.

Now the cosmids enter into bacterial cell and replicates as usual plasmids since
they are having bacterial origin.

Cosmids provide an efficient means of cloning large pieces of foreign DNA

(35- 45 Kb).

Because of their capacity for large fragments of DNA, cosmids are particularly
attractive vectors for constructing libraries of eukaryotic genome fragments.
Phasmids (or Phagemids)

These are plasmid vectors having both bacterial and phage origin of replication
Example pBluescript II KS
M-13 phage based cloning vectors have the follosing disadvantages
1. difficult to obtain souble strand DNA
2.Roling circle replication is affected by large size of insert
3. Low yield of Recombinant DNA

To overcome these problems hybrid of M13 phage and plasmid cloning vector
were constructed.

The original phagemid vector is derived from pUC18 and pUC19

Multiple cloning sites

Lac Z selection system
Fl origin of replication for both + and - strands
Col E1 ori for plasmid replication in E.coli.
Ampicilin antibiotic marker gene

Viral vectors for animal cells

viruses can deliver their nucleic acid into cells and the high levels of replication
and gene expression it is possible to achieve.

Viruses have been used as vectors not only for gene expression in cultured cells
but also for gene transfer to living animals.

For viral vectors, the usual approach is to remove the unneeded or pathogenic
features while retaining the efficiency of gene delivery, expression and
persistence
where appropriate.

Properties of viral vectors

Viral vectors are tailored to their specific applications but generally share a few
key properties.

Safety: Although viral vectors are occasionally created from pathogenic viruses,
they are modified in such a way as to minimize the risk of handling them.

This usually involves the deletion of a part of the viral genome critical for viral
replication. Such a virus can efficiently infect cells but, once the infection has
taken place, requires a helper virus to provide the missing proteins for
production of new virions.
If the transgene is added to the genome or if it replaces one or more genes that
are non-essential for the infection cycle in the expression host being used, the
vector is described as replication-competent or helper-independent, because it
can propagate independently.

However, if the transgene replaces an essential viral gene, this renders the
vector replication-defective or helper-dependent, so that missing functions must
be supplied in trans.

This can be accomplished by cointroducing a helper virus or transfecting the

cells
with a helper plasmid, each of which must carry the missing genes

Low toxicity: The viral vector should have a minimal effect on

the physiology of the cell it infects.

Stability: Some viruses are genetically unstable and can rapidly rearrange their
genomes. This is detrimental to predictability and reproducibility of the work
conducted using a viral vector and is avoided in their design.

Cell type specificity: Most viral vectors are engineered to infect as wide a range
of cell types as possible. However, sometimes the opposite is preferred. The
viral receptor can be modified to target the virus to a specific kind of cell.
Viruses modified in this manner are said to be pseudotyped.

Identification: Viral vectors are often given certain genes that help identify
which cells took up the viral genes. These genes are called Markers, a common
marker is antibiotic resistance to a certain antibiotic.

Four classes of viral vector have been developed for use in human gene therapy
and have reached phase 1 clinical trials. These are
Retrovirus,
Adenovirus,
Herpesvirus and
Adenoassociated virus (AAV)

Adenovirus
Adenoviruses are DNA viruses with a linear, doublestranded genome of
approximately 36 kb.

There are six early-transcription units, most of which are essential for viral
replication, and a major late transcript that encodes components of the capsid.

Adenoviruses have been widely used as gene transfer and expression vectors,
because they have many advantageous features, including stability, a high
capacity for foreign DNA, a wide host range that includes non-dividing cells,
and the ability to produce high-titre stocks (up to 1011 plaque-forming units
(pfu)/ml).

They are suitable for transient expression in dividing cells because they do not
integrate efficiently into the genome, but prolonged expression can be achieved
in post-mitotic cells, such as neurons.

Adenoviruses are particularly attractive as gene therapy vectors, because the

virions are taken up efficiently by cells in vivo and adenovirus-derived vaccines
have been used in humans with no reported side-effects.

However, the recent death of a patient following an extreme inflammatory

response to adenoviral gene-therapy treatment underlines the necessity for
rigorous safety testing

Most early adenoviral vectors were replication deficient, lacking the essential
E1a and E1b genes and often the non-essential gene E3.

These first generation E1 replacement vectors had a maximum capacity of

about 7 kb and were propagated in the human embryonic kidney line 293

Although these vectors have been used with great success, they suffer from two
particular problems:
cytotoxic effects, resulting from low-level expression of the viral gene products,
and the tendency for recombination to occur between the vector and the
integrated portion of the genome, resulting in the recovery of replication-
competent viruses.

Higher-capacity vectors have been developed, which lack the E2 or E4 regions

in addition to E1 and E3, providing a maximum cloning capacity of about 10
kb.

Adeno-associated virus
AAV is not related to adenovirus, but is so called because it was first discovered
as a contaminant in an adenoviral isolate.

AAV is a single-stranded DNA virus, a member of the parvovirus family, and is

naturally replication defective, such that it requires the presence of another virus
(usually adenovirus or herpesvirus) to complete its infection cycle.

In adenovirus- or herpesvirus-infected cells, AAV replicates lytically and

produces thousands of progeny virions.

However, in the absence of these helpers, the AAV DNA integrates into the
host cells genome, where it remains as a latent provirus.

In human cells, the provirus integrates predominantly into the same genetic
locus on chromosome 19. Subsequent infection by adenovirus or herpesvirus
can rescue the provirus and induce lytic infection.

Advantages include the wide host range, which encompasses non-dividing cells
The AAV genome is small (about 5 kb) and comprises a central region
containing rep (replicase) and cap (capsid) genes flanked by 145-base inverted
terminal repeats.

In the first AAV vectors, foreign DNA replaced the cap region and was
expressed from an endogenous AAV promoter

The AAV genome is small (about 5 kb) and comprises a central region
containing rep (replicase) and cap (capsid) genes flanked by 145-base inverted
terminal repeats.

In the first AAV vectors, foreign DNA replaced the cap region and was
expressed from an endogenous AAV promoter

AAV vectors have been used to introduce genes efficiently into many cell types,
including liver, muscle and neurons. However, deletion of the rep region
abolishes the site specificity of proviral integration, so the vector integrates at
essentially
random positions, which may increase the risk of insertional gene inactivation

The fact that AAV uses concatemeric replication intermediates has been used to
circumvent perhaps the most serious disadvantage of AAV vectors, which is the
limited capacity for foreign DNA.

Baculovirus
Baculoviruses have rod-shaped capsids and large, dsDNA genomes. They
productively infect arthropods, particularly insects.

One group of baculoviruses, known as the nuclear polyhedrosis viruses, have an

unusual infection cycle that involves the production of nuclear occlusion
bodies.

These are proteinaceous particles in which the virions are embedded, allowing
the virus to survive harsh environmental conditions, such as desiccation.

Baculovirus vectors are used mainly for high-level transient protein expression
in insects and insect cells.

The occlusion bodies are relevant to vector development because they consist
predominantly of a single protein, called polyhedrin, which is expressed at very
high levels.

The nuclear-occlusion stage of the infection cycle is non-essential for the

productive infection of cell lines; thus the polyhedrin gene can be replaced with
foreign DNA, which can be expressed at high levels under the control of the
endogenous polyhedrin promoter.

Two baculoviruses have been extensively developed as vectors, namely the

Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and the
Bombyx mori nuclear polyhedrosis virus (BmNPV).

The former is used for protein expression in insect cell lines, particularly those
derived from Spodoptera frugiperda The latter infects the silkworm and has
been used for the production of recombinant protein in live silkworm larvae

Polyhedrin gene-replacement vectors are the most popular due to the high level
of recombinant protein that can be expressed (up to 1 mg/106 cells)

region are important for

high-level foreign gene expression and these are included in all polyhedrin
replacement vectors

wild-type viruses cause the microscopic viral plaques to appear opalescent if

viewed under plaques appear
clear (OB)
Insertion of the E. coli lacZ gene in frame into the polyhedrin coding region
allows bluewhite screening of recombinants in addition to the OB assay

Although baculoviruses productively infect insect cells, they can also be taken
up by mammalian cells, and this suggests that recombinant baculoviruses could
be developed as vectors for gene therapy

More recently, cell lines have been generated that are stably transformed with
baculovirus vectors

Retrovirus vectors

Retroviruses are RNA viruses that replicate via a dsDNA intermediate. The
infection cycle involves the precise integration of this intermediate into the
genome of the host cell, where it is transcribed to yield daughter genomes that
are packaged into virions.

Immediately after infection, the RNA genome is reverse transcribed to produce

a cDNA copy.

The DNA intermediate then integrates into the genome at an essentially random
site

Retroviruses are acutely oncogenic because they carry particular genes that
promote host cell division.

Retroviruses act as replication defective gene-transfer vectors

Most retroviruses do not kill the host, but produce progeny virons over an
indefinite period. Retroviral vectors can therefore be used to make stably
transformed cell lines

Viral gene expression is driven by strong promoters, which can be subverted to

control the expression of transgenes

Murine leukaemia virus (MLV), have a broad host range, allowing the
transduction
of many cell types. Finally, retroviruses make efficient and convenient vectors
for gene transfer because the genome is small enough for DNA copies to be
manipulated in vitro in plasmid cloning vectors

The major disadvantage of oncoretroviral vectors is that they only productively

infect dividing cells, which limits their use for gene-therapy applications
Herpesvirus vectors

The herpesviruses are large dsDNA viruses that include HSVs (e.g. HSV-I,
varicella zoster). Most HSVs are transmitted without symptoms (varicella zoster
virus is exceptional) and cause prolonged infections.

Viral replication can occur in many cell types in a wide range of species if the
genome is introduced by transfection, but HSV vectors are particularly suitable
for gene therapy in the nervous system, because the virus is remarkably
neurotropic.

Therapeutic use of herpesvirus vectors has been limited, but a number of genes
have been successfully transferred to neurons in vivo

Note that HSVis also transmitted across neuronal synapses during lytic
infections, a phenomenon that can be exploited to trace axon pathways
Binary vector. In this system, the T-DNA and the vir region reside in separate plasmids within
the same Agrobacterium strain. The vir genes are located in a disarmed (without tumor genes)
Ti plasmid and the T-DNA with the gene of interest is located in a small vector molecule.

Co-integrated vector. It results from the recombination of a small vector plasmid, for example
an E.
coli vector, and a Ti plasmid harbored in A. tumefaciens . The recombination takes place
through a
homologous region present in both of the plasmids. An engineered T-DNA containing the gene of
interest can be in either one of the plasmids.

Binary vectors
The discovery that the vir genes do not need to be in the same plasmid with a T-DNA region to
lead its transfer and insertion into the plant genome led to the construction of a system for plant
transformation where the T-DNA region and the vir region are on separate plasmids.
In the binary vector system, the two different plasmids employed are:
a wide-host-range small replicon , which has an origin of replication (ori) that permits the
maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium .
This plasmid typically contains:
1. foreign DNA in place of T-DNA,
2. the left and right T-DNA borders (or at least the right T-border),
3. markers for selection and maintenance in both E. coli and A. tumefaciens ,
4. a selectable marker for plants.
The plasmid is said to be "disarmed", since its tumor-inducing genes located in the T-DNA have
been removed.
a helper Ti plasmid, harbored in A. tumefaciens , which lacks the entire T-DNA region but
contains an intact vir region.

In general, the transformation procedure is as follows:

the recombinant small replicon is transferred via bacterial conjugation or direct transfer to
A. tumefaciens harboring a helper Ti plasmid,
the plant cells are co-cultivated with the Agrobacterium , to allow transfer of recombinant T-DNA
into
the plant genome, and transformed plant cells are selected under appropriate conditions.

Possible pitfalls
A possible disadvantage may ensue from the fact that the stability of wide host range replicons in
E. coli and Agrobacterium varies considerably. Depending on the orientation, plasmids with two
different origins of replication may be unstable in E. coli where both origins are active.
Advantages
Compared with co-integrated vectors, binary vectors present some advantages:
No recombination process takes place between the molecules involved.
Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which
increases
transfer efficiency from E. coli to Agrobacterium .
This vector system is most widely used nowadays. Different types of binary vectors have been
devised to suit different needs in a plant transformation process.
Binary vector types
pGA series vectors, pCG series vectors, pBECK2000 series, pGreen series,

Co-integrated vectors
Called co-integrated vectors or hybrid Ti plasmids, these vectors were among the first types of
modified and engineered Ti plasmids devised for Agrobacterium -mediated transformation, but
are not widely used today.
These vectors are constructed by homologous recombination of a bacterial plasmid with the T-
DNA region of an endogenous Ti plasmid in Agrobacterium . Integration of the two plasmids
requires a region of homology present in both.

Three vectors are necessary in this system:

Disarmed Agrobacterium Ti plasmids
In these Ti plasmids, the oncogenes located in the T-DNA region have been replaced by
exogenous
DNA.
Examples of these vectors include:
SEV series: the right border of the T-DNA together with the phytohormone genes coding for
cytokinin and auxin are removed and replaced by a bacterial kanamycin resistance gene while
the
left border and a small part of the left segment (TL) of the original T-DNA (referred to as Left
Inside Homology (LIH)) are left intact.
1.
pGV series: the phytohormone genes are excised and substituted by part of pBR322 vector
sequence. The left and right border sequences as well as the nopaline synthase gene of the Ti
plasmid are conserved.

A resulting co-integrated plasmid assembled by in vitro manipulation normally contains:

1. the vir genes,
2. the left and right T-DNA borders,
3. an exogenous DNA sequence between the two T-DNA borders, and
4. plant and bacterial selectable markers.
Some drawbacks
Although co-integrated vectors have been designed to allow site-specific recombination based on
the recombination system of the phage P1 (e.g., wP1lox P-Cre series), co-integrated vectors in
general are less popular due to:
long homologies required between the Ti plasmid and the E. coli plasmids making them difficult
to
engineer and use, and relatively inefficient gene transfer compared to the binary vectors.

Ti plasmid

The plant disease, crown gall, which was observed by Aristotele

and His follower Theophrostus
 Smith and Townsend 1907, studied the Galls of cultivated Paris
daisy and for the first time established that this plant tumour
is of bacterial origin( Bacterium tumefaciens).
During the next thirty years the name of the bacterium was
transformed from Bacterium tumefaciens through Phytomonas
tumefaciens, Bacillus tumefaciens to Agrobacterium
tumefaciens
Sehilperoot et al.(1967). The nature of tumourous state is
decided by the genotypic nature of Agrobacterium strain.
Johnson et al.,1973. the information for synthesis of opine
resides in the plant genome.
Montaya et al., 1973 later on accepted the mistake
Braun 1941 1969. It was believed that unlike animal tumours
Crown gall required constant presence of bacteria.
But Braun showed that presence of bacteria is not required for
secondary crown gall production. He along with White (1941)
found that unlike normal plant tissue crown gall tomour tissue
could proliferate in auxin free white basal medium.

1). Agrobacteria will induce crown gall only after the wounding
of the plant,

2). The Agrobacteria produce a tumourigenic principle (Ti

plasmid) that is essential for future tumour development,
Ti Plasmid causes reorientation of the metabolism in the host
cells
4).Once this reorientation takes place there is no turning back,
5).Once the transformed tumor cells are autonomous in their
ability of perpetual cell division , and
6).The transformation induced synthesis of auxin and
cytokinin is related to the perpetual cell division
1974 is the most important year for Agrobacterium research.
During this year it was established that Ti plasmid is
responsible for tumour development.
Hence Hooykaas suggested that Ti plasmid can be used as a
vector.
 Complete Ti-plasmid DNA is not found in plant tumor cells but a
small, specific segment of the plasmid, about 23 kbp in size, is
found integrated in the plant nuclear DNA
This DNA segment is called T-DNA (transferred DNA) and
carries genes for growth and opine synthesis
T-DNA comprises two segments: TL (which carries the genes
required for tumour formation) and TR (which carries the genes
for opine synthesis).
The two segments are transferred to the plant genome and
may be present as multiple copies.
T-DNA is flanked by 25 bp imperfect direct repeats known as
border sequences, which are conserved between octopine and
nopaline plasmids.
The border sequences are not transferred intact to the plant
genome, but they are involved in the transfer process
The genes responsible for T-DNA transfer are located in a
separate part of the Ti plasmid called the vir (virulence) region.
 Vir A gene acts as the receptor for certain phenolic molecules
that are released by wounded plant cells.
Activated VirA transphosphorylates (induces) the VirG protein
In addition to virG, further genes on the bacterial chromosome
also encode transcription factors that regulate vir gene
expression
The components of the pilus are encoded by genes in the virB
operon
DNA transfer itself is initiated by an endonuclease formed by
the products of the virD1 and virD2 genes.
This introduces either single-strand nicks or a double-strand
break at the 25 bp borders of the T-DNA, a process enhanced
by the VirC12 and VirC2 proteins
The VirD2 protein remains covalently attached to the processed
T-DNA.
T strands are coated with VirE2, a single-stranded DNA-binding
protein.

Cointegrated and Binary vector system

These vectors are constructed by homologous recombination

of a bacterial plasmid with the T-DNA region of an endogenous
Ti plasmid in Agrobacterium. Integration of the two plasmids
requires a region of homology present in both.
co-integrated plasmid assembled by in vitro manipulation
normally contains:
the vir genes,
the left and right T-DNA borders, an exogenous DNA sequence
between the two T-DNA borders, and plant and bacterial
selectable markers.
Binary vector

The discovery that the vir genes do not need to be in the same
plasmid with a T-DNA region to lead its transfer and insertion
into the plant genome led to the construction of a system for
plant transformation where the T-DNA region and
the vir region are on separate plasmids.
In the binary vector system, the two different plasmids
employed are:
a wide-host-range small replicon contains:
foreign DNA in place of T-DNA,
the left and right T-DNA borders (or at least the right T-
border),
markers for selection and maintenance in both E.
coli and A. tumefaciens,
a selectable marker for plants

a helper Ti plasmid, harbored in A. tumefaciens, which lacks the

entire T-DNA region but contains an intactvir region.
 Virus mediated gene transfer

Many virus infections are systemic so that gene can be introduced into all cells
in a plant.

Virus infections results in the addition of new genetic material. Additional

genetic material incorporated into the genome of plant virus might be replicated
and expressed in plant cell.

once biological characters of virus have been suitably selected and manipulated
new genes must be incorporated into virus in such a way that they are expressed
in the plant
The replicating genomes of plant viruses are non integrative vectors as
compared to Agrobacterium vectors

The viral vectors are non integrative vectors (do not integrate their DNA in to
the host genome) rather they spread systematically within a plant and
accumulate high copy numbers.

Apart from their pathogenicity recombinant viruses are designed to mimic their
wild type counterparts in all other respects.
Viral vector should posses the following characteristics

1. It should have broad host range, virulence, ease of mechanical transmission.

2. It should have the potential to carry additional genetic information

3.Virus suitability as a vector depends on the fact that genetic material must be
able to be manipulated and be infectious.

4. should be transmitted mechanically because their genomes are often

infectious However, some viruses are not amenable to mechanical inoculation.
For example, phloem limited luteoviruses and geminiviruses are recalcitrant
to mechanical passage from plant to plant.

Advantage of viral vectors

Because of the nature of virus infections, the maximum level of foreign gene
expression from viral genomes is predicted to occur within a short time-period,
usually within one or two weeks after inoculation

Another practical advantage is that many plant viruses are easily transmissible
and could potentially be used commercially for rapid mechanical inoculation
of large acreages of crop plants

This strategy has the potential to expedite initial screening procedures for
selection by testing the possible effects of mutated genes before implementing
more time and resource consuming transgenic approaches for genetic analyses
or breeding

Caulimoviruses

The caulimovirus is the most potent plant virus

Their genome is double stranded DNA which is easy to manipulation
They are the first virus among the manipulated virus
There are 19 viruses with limited host range
The best known member is CaMV Cauliflower Mosaic Virus
Naturally transmitted by aphids and easily be transmitted mechanically
High copy number in cells 106 per cell
within 3-4 weeks it infects the entire plant
The genome is relaxed circular DNA around 8 kb ORF VIII can be removed
and gene of interest may be inserted

Advantage
Naked DNA is infective
More suited for experiments

Disadvantage
Tightly packed and little space for insertion
Has multiple cleavage sites- tough for nmanipulation

Gemini viruses

Geminiviruses cause disease in wide varieties of plants

Curly Top Virus (CTV) and Maize Streak Virus (MSV).
Covalently closed circular DNA, 2.6-3Kb single stranded
The genomic SS DNA replicates in the nucleus through double stranded DNA

RNA viruses
RNA viruses have highest level of expression
e.g tobacco mosaic virus
size : 270bp- 1.5kb
Disadvantage of viral vectors
Development of symptoms, choice is less, very short size inserts
Direct DNA transfer
Particle bombardment refers to a method where heavy-metal particles (1 mm
gold or tungsten) are coated with DNA, accelerated toward the target tissue, and
penetrate the cell wall to rest either adjacent to or directly in the nucleus.

The DNA on the particles somehow finds its way to the native DNA of the
target cell, where it becomes integrated into the chromosome to become a
permanent addition to the genome.

The term particle bombardment can be used interchangeably with the similar
terms
microprojectile bombardment, Biolistics, particle acceleration, and gene gun
technology.
The term that is currently most often used is particle bombardment.

As opposed to Agrobacterium, which is a biological vector for DNA

introduction, particle bombardment is a purely physical method for DNA
delivery.

The DNA is physically precipitated onto metal particles, and those particles are
then rapidly accelerated toward the target tissue.

The particles penetrate through the cell wall by punching holes in that rigid
structure and continue to do so until being stopped by the density of the target
tissue.

Particle bombardment was invented by John Sanford and colleagues in the mid-
1980s.

Today, in most laboratories, high-pressure helium is used to generate the force

needed to accelerate small gold particles toward the target tissue.

DNA is first precipitated onto the particles, which are then placed as a
monolayer on a Mylar carrier sheet, called a macrocarrier (this term refers to the
structure that carries the particles, while microcarrier was the term originally
designated for the particles, as they are small and carry the DNA).

The controlled explosion used to accelerate the macrocarrier is provided by

high-pressure helium, which is released from a small chamber following the
breakage of a ruptured disk, designed to break at specific pressures.

The macrocarrier, with the particles on one side, travels a short distance and
smashes into a screen, stopping the macrocarrier and allowing the particles to
continue along their path.

In most cases, the whole procedure is performed under partial vacuum, because
the presence of air slows down the particles. A partial vacuum, applied for a
short duration, does not appear to damage the biological targets.
Advantages
As particle bombardment is a physical method for DNA introduction,
complications from biological interactions with the plant (as with
Agrobacterium-mediated transformation) are avoided.

A wide variety of plant tissues can be used as targets for particle bombardment.
These range from embryos, seedlings, shoot apices, leaf disks, microspores, and
immature pollen grains to potato tubers and nodes.

Although the foreign DNA integration patterns (discussed above) can be very
complex, this mechanism for DNA recombination and integration can be an
advantage.

Various DNAs can be mixed and co-introduced using a method called co-
transformation. Reports of 1215 different DNAs have been successfully co-
transformed into soybean

Particle bombardment remains the only method that can be used for
transformation of chloroplasts and mitochondria.

Disadvantages
The main perceived limitations are the randomness of DNA integration and the
high copy number of introduced DNAs. As with most methods of DNA
introduction, the position and orientation of the transgene in the plant
chromosome will differ with every transformation event.

The location of the transgene within the target chromosome will influence the
expression of that gene. Transgenes in more active regions of genomic DNA
will express at higher levels, while integration in less active areas will lead to
lower expression: position effects.

Transient expression the expression of transgene remains for short time

because it did not integrate in to genome
Deterioration of regeneration potentiality of target tissues due to high wound
created by bombardment.
Cost of the instrument and gold particles are high.

Electroporation
Electroporation is the application of strong electric field pulses to cells and
tissue is known to cause some type of structural rearrangement of the cell
membrane resulting in a temporary increase in porosity and providing a local
driving force for ionic and molecular transport through the pores.
In vitro introduction of DNA into cells is now the most common application of
electroporation. Several physical factors such as created transmembrane
potential by the imposing pulse electric field, exent of membrane permeation,
duration of the permeated state, mode and duration of molecular flow, global
and local (surface) concentrations of DNA, form of DNA, tolerance of cells to
membrane permeation and the heterogeneity of the cell population may affect
the electroporation efficiency.
It uses 1-1.5mv with low capacitance and therefore a short decay time
The chamber is cylindrical in form with a distance of 2cm between parallel steel
electrodes
Electroporation has several advantages over biolistics in that it does not require
the expensive particle gun apparatus, associated consumable supplies and
licensing and has worked well for stable-transformation experiments.
Linear DNA can be transferred in this method
Simple, Fase and low toxicity to cells are the other advantages.
The range of tissues that can be transformed by electroporation seems to be
narrower.
Microinjection
Transformation via microinjection is based on introducing DNA into the
nucleus or cytoplasm by means of a glass micro capillary-injection pipette.

This method is proposed to transfer organells and for the manipulation of

isolated chromosomes

This operation requires a micromanipulator. During the introduction of DNA

into the nucleus, cells are immobilized with a holding pipette and gentle suction.

When cells or protoplasts are used as targets in this technique glass

micropipettes of 0.5-10m tip (needle like) are used for transfer of DNA

Both pipettes contain mineral oil, which works as a cylinder. Microinjection is

mainly used for the transformation of large animal cells. Its importance
for plant transformation is rather limited due to the characteristics of plant cell
walls, which contain a thick layer of lignins and cellulose. The plant cell wall is
a barrier for glass micro tools.
The microinjection of protoplast could theoretically resolves this limitation, but
it carries with it the danger of releasing of hydrolases and other toxic
compounds from the vacuole to the cytoplasm, which can cause rapid death of
the protoplast.

The microinjection of protoplasts requires different methods of

immobilizationinstead of using a sucking capillary; protoplasts are attached
to glass by coating them by poly-L-lisine or agarose. None of these solutions has
proved useful, as poly-L-lysine can be toxic for some species and agarose (even
a very thin layer) reduces visibility in the area of manipulation.

Currently, microinjection is widely used for the transformation of large animal

cells, whereas it has not been developed into a routine transformation method
for plants. The procedure is very slow and requires an expensive
micromanipulator. However, one of the unquestionable improvements of
microinjection was that it allowed the introduction not only of DNA plasmids
but also of whole chromosomes into plant cells.
Fig.3 Microinjection
Polyethylene Glycol Mediated (PEG) Transformation
The most common method of delivering foreign DNA into plant protoplasts
involves treatment with PEG as a hydrophilic long-chain polymer.
In this method isolated protoplast are taken in a tube followed by addition of
plasmid DNA . To this 40% PEG dissolved in manitol is added.
Many cells can be handled with ease, many independent transformants can be
produced, the selection of transformants is relatively simple and no specialized
equipment is needed. As mentioned earlier, regenerating whole plants from
transgenic protoplasts has proven difficult.
The system is only rarely employed due to low frequency of transformants and
the inability of many species to be regenerated into whole plants from
protoplasts.

Liposome mediated transfection (Lipofection)

A derivation of PEG mediated transformation is the liposome mediated
transformation technique.
Cationic liposomes are positively changed lipids and are increasingly used for
DNA uptake due to their favorable interactions with negatively charged DNA
and cell membranes.
In this approach foreign DNA must be encapsulated in a spherical lipid bilayer
termed a liposome to prepare lipoplexes.
In this method DNA enters protoplast due to endocytosis of liposomes and this
involves following steps
1. Adhesion of protoplast to the protoplast surface
2. Fusion of liposomes at the site of adhesion
3. Release of plasmid inside the cell
After endo cytosis the DNA is free to recombine and integrate into the host
genome. Successful transformation based on this system was reported for
tobacco
The encapsulation of DNA protects from nuclease digestion
Reduced level of cell toxicity
High degree of reproducibility.
Silicon carbide fibers (whiskers)
One of the recently developed methods to delivery of DNA to plants is Silicon
Carbide Mediated transformation (SCMT). Physical and chemical
characteristics of silicon carbide fibres make them capable of puncturing the
cells without killing them.

Silicon carbide whiskers are long, rigid two-pointed microscopic spears that
are added to plant cells and DNA and then vortexed. The spears or whiskers are
approximately 1 mm thick and 1550 mm long.

The advantage of this system is: rapid, inexpensive, easy to set-up and is
effective on a variety of cell types.

Disadvantage : The low efficiency of transformation using silicon carbide

whiskers along with disposal under conditions similar to those for asbestos
render this method unsuitable for most laboratories.

The efficiency of SCMT depends on the fiber size, parameters of vortexing,

shape of the vessel used, plant species and explants and characteristics of the
plant cells and especially thickness of cell wall.

Ultrasound mediated transformation

Ultrasound is used to described for stimulating uptake of foreign DNA by plant
protoplasts and leaf segments of tobacco.
This procedure involves immersion of explants in sonication buffer containing
plasmid DNA.
Sonication with ultrasonicator at 0.5 w/cm for 30 min. and grow the plants
Advantage : No need for tissue culture. Disadvantage : low success rate.

Nanofiber Arrays
Successful use of nanofiber arrays (Melechko et al. 2005) for DNA introduction
into plant cells

Nanofiber arrays can best be described as a microscopic bed of nails

Early attempts to generate nanofiber arrays resulted in the formation of

nanoscale pyramid-shaped structures on a silicon chip

In this early work, the surface of the chips was precisely etched away, to leave
the nanofiber pyramids. The newer arrays are composed of long, thin structures,
and they hold much more promise for success with DNA introduction into plant
cells.

Nanofiber arrays are actually grown on chips, with very precise composition,
height, and spacing possible. DNA can be chemically bound to the fiber or
simply precipitated onto it.

For successful DNA introduction into animal cells, the arrays were stationary
and the animal/ plant cells were propelled toward the chip. Cells were then
allowed to grow.

UNIT III CLONE IDENTIFICATION

Introducing DNA in to living cells

The next step in a gene cloning experiment is to introduce these molecules into
living cells, usually bacteria, which then grow and divide to produce
clones

Strictly speaking, the word cloning refers only to the later stages of the
procedure, and not to the construction of the recombinant DNA molecule itself.

a large number of recombinant DNA molecules to be produced from a limited

amount of starting material.

At the outset only a few nanograms of recombinant DNA may be available, but
each bacterium that takes up a plasmid subsequently divides numerous times to
produce a colony, each cell of which contains multiple copies of the molecule.

colony is used not as a source of DNA but as an inoculum for a liquid culture,
the resulting cells may provide milligrams of DNA, a millionfold increase in
yield. In this way cloning can supply the large amounts of DNA needed for
molecular biological studies of gene structure and expression

ligation mixture may contain, in addition to the desired recombinant molecule,

any
number of the following:
Unligated vector molecules, Unligated DNA fragments
Vector molecules that have recircularized without new DNA being inserted
(self-ligated vector)
Recombinant DNA molecules that carry the wrong inserted DNA fragment.
Unligated molecules rarely cause a problem

Transformationthe uptake of DNA by bacterial cells

Most species of bacteria are able to take up DNA molecules from the medium in
which they grow.

Often a DNA molecule taken up in this way will be degraded, but occasionally
it
is able to survive and replicate in the host cell. In particular this happens if the
DNA molecule is a plasmid with an origin of replication recognized by the host.

In nature, transformation is probably not a major process by which bacteria

obtain
genetic information.

only a few species (notably members of the genera Bacillus and Streptococcus)
can be transformed with ease.
Most species of bacteria, including E. coli, take up only limited amounts of
DNA
under normal circumstances. In order to transform these species efficiently, the
bacteria have to undergo some form of physical and/or chemical treatment that
enhances their ability to take up DNA. Cells that have undergone this treatment
are said to be competent.

Preparation of competent E. coli cells

it was observed that E. coli cells that had been soaked in an ice cold salt
solution were more efficient at DNA uptake than unsoaked cells.

A solution of 50 mM calcium chloride (CaCl2) is traditionally used, although

other salts, notably rubidium chloride, are also effective.

Possibly CaCl2 causes the DNA to precipitate onto the outside of the cells, or
perhaps the salt is responsible for some kind of change in the cell wall that
improves DNA binding.

The actual movement of DNA into competent cells is stimulated by briefly

raising the temperature to 42C. Once again, the exact reason why this heat
shock is effective is not understood.

Selection for transformed cells

Transformation of competent cells is an inefficient procedure, however
carefully the cells have been prepared.

Although 1 ng of the plasmid vector called pUC8 can yield 100010,000

transformants, this represents the uptake of only 0.01% of all the available
molecules.
E. coli cells are normally sensitive to the growth inhibitory effects of the
antibiotics ampicillin and tetracycline.

However, cells that contain the plasmid pBR322 which was one of the first
cloning vectors to be developed back in the 1970s, are resistant to these
antibiotics.

E. coli cells that have taken up a plasmid are ampRtetR and able to form
colonies on an agar medium. Transformants and non-transformants are therefore
easily distinguished.

Molecular mechanism of T-DNA transfer

For T- DNA transfer a segment of Ti plasmid 40 kb called the virulence region
(vir genes) and the hromosome (chv genes) are required

The chvA and chvB genes are necessary for the attachment of Agrobacterium to
plant cell walls .

The chvB gene codes for a 235 kDa protein involved in the formation of a cyclic
-l,2 glucan The chvA gene determines a transport protein located in the
bacterial inner membrane necessary for the transport of the fi-1,2 glucan into
the periplasm

The 40 kb vir region of the octopine Ti plasmid embraces 24 genes involved in

virulence. These genes are present in 8 operons called virA-virH, which are co-
regulated and thus form a regulon.

The early steps in plant tumour induction concerns the coordinate activation of
the virulence system, when the bacteria are present near (wounded) plant tissues
and sense plant cell exudate factors

chv genes are constitutively expressed, the vir genes are silent until they become
induced by certain plant factors.

With the exception of the virA and virG genes, the vir operons are not
transcribed during normal vegetative growth

Stachel identified these plant factors from tobacco as being the phenolic
compounds acetosyringone and ~-hydroxyacetosyringone. These compounds
are released from plant tissue, especially after wounding, which has been long
known to be a prerequisite for plant tumorigenesis via Agrobacterium.
Recently, it was found that some specificity exists in the sugars that are required
for optimal vir induction( 2-deoxyd- glucose and 6-deoxy-d-glucose have such a
stimulatory effect.

Two proteins encoded by the virulence region, VirA and VirG, mediate the
activation of the other vir genes in the presence of phenolic inducers

VirA-like proteins are sensors for a specific signal (phenolic compounds in the
case of VirA), while the VirG-like proteins are DNA-binding activator proteins.

VirA protein is present in the bacterial inner membrane. Acetosyringone is a

lipophilic compound that easily would accumulate in the bacterial inner
membrane or pass through it

The formation of both ss- and ds-T molecules is dependent on the activity of
two proteins called VirD 1 and VirD2 that are encoded by the virD operon of
the vir region

These proteins together determine an endonuclease activity capable of nicking

(introducing ss breaks) the border repeats at a precise site

It is likely that the nick sites act as starting points for DNA synthesis in the 5' ~
3' direction. T-strands will then be released by displacement

Deletion of the right-border repeat almost completely abolished T-DNA transfer

The introduction of nicks at border repeats by the VirD system followed by ss

T-strand formation
ssDNA is quickly converted into dsDNA in plant cells.

T-strands retain the VirD2 protein covalently attached to the 5' terminus. The
presence of VirD2 makes the 5' end of the T-strand less vulnerable to an attack
by exonucleases

VirD2 protein may act as a pilot to direct the T strand to the nucleus of the
transformed plant cell, since it contains nuclear targeting sequences

The 69 kDa VirE2 protein encoded by the second open reading frame (orf) of
the virE operon is a ssDNA-binding protein, which is able to coat the T-strands
by cooperative binding leading to long, thin nucleo-protein filaments

In bacteria ssDNA is transferred from donor to recipient via a conjugative pore

(encoded by Tra proteins)

VirC 1 protein specifically binds to the overdrive sequence.- product of the

virC1 gene, which binds to the T-DNA transfer enhancer.

Above the roles played by VirD, VirC and VirE proteins in T-complex
formation are described in detail, as well as the way vir expression is regulated
via VirA and VirG. The remaining Virproteins are not involved in regulation of
expression or T-strand formation; only those encoded by the virB operon are
essential for virulence.

Vir B proteins together (11 genes) may form a structure (conjugal pore or pilus)
through which the T-DNA is delivered into the plant cell. The virB operon
determines a transfer apparatus similar to that of conjugative plasmids
The virH operon consists of two genes that code for proteins that show some
similarity to cytochrome P450 enzymes. These proteins may therefore have a
role in the detoxification of certain plant compounds that might otherwise
adversely affect the growth of Agrobacterium.

virF that is needed for complementation for tumor formation.

Maxam & Gilbert Sequencing

There are four chemical cleavage reactions at the core of the Maxam and Gilbert
sequencing system. The figure below left shows an example from these reactions, the
reaction cleaving specifically at guanine. The other three reactions cleave at G+A, C+T,
or C. Guanine and cytosine, therefore, give bands in 2 lanes, adenine and thymine in only
one. An example of the gel pattern produced is presented below right.

In a Maxam and Gilbert gel, the identity of guanine or cytosine

Maxam and Gilbert DNA sequencing reaction specific for Guanidine residues.
The Guanine base is first modified with Dimethyl Sulfate (DMS), which makes the
chain susceptible to cleavage by piperidine, destroying the Guanidine residue
and releasing a labeled fragment for electrophoresis.
in the sequence can be assigned most easily because two of the
four reaction sets cleave at those bases alone. Adenine or
thymine are slightly more difficult, being represented by those
bands in the G+A or C+T lanes which do not appear,
respectively, in the G or C lanes.

The DNA to be sequenced must first be end labeled, at one end only. This is
accomplished by kinase treatment with 32P ATP, which labels both ends, followed by
restriction digestion and isolation of the two labeled fragments. Alternatively, digestion
of a plasmid containing a clone of the DNA of interest with an appropriate enzyme can
yield a unique labeling site. Plasmid vectors containing the rare site for Tth111I, which
leaves a single 5' base overlap, have been generated for this purpose. Cleavage with
Tth111I leaves a G at one end and a C at the other in these vectors. By filling in the gap
with Klenow polymerase fragment in the presence of dGTP or dCTP, one end or the
other can be labeled specifically. Labeled DNA is first precipitated to remove any salts
which might interfere in the cleavage reactions. It is then modified, cleaved and run on a
denaturing gel for analysis. NB: THE HYDRAZINE AND DMS USED IN THESE
PROTOCOLS ARE TOXIC AND VOLATILE. KEEP TUBES SEALED AND WORK IN A
HOOD
Piperidine is used for G and for AG acid used after Piperidine For C
hydrazine is used and for CT with acid used
Methods for clone identification

After cloning all the possible genes from an organism into a library, the next
step is to identify the gene of interest.

A number of procedures can be employed to attempt identification of the

desired clone.

Although a few of these procedures are based on detection of the translation

product of the cloned gene, it is usually easier to identify directly the correct
recombinant DNA molecule.

This can be achieved by the important technique of hybridization probing

Any two single-stranded nucleic acid molecules have the potential to form base
pairs with one another. With most pairs of molecules the resulting hybrid
structures are unstable, as only a small number of individual interstrand bonds
are formed.

However, if the polynucleotides are complementary, extensive base pairing can

occur to form a stable double-stranded molecule Nucleic acid hybridization
can be used to identify a particular recombinant clone if a DNA or RNA probe,
complementary to the desired gene, is available

Colony and plaque hybridization probing

Hybridization probing can be used to identify recombinant DNA molecules

contained in either bacterial colonies or bacteriophage plaques.

First the colonies or plaques are transferred to a nitrocellulose or nylon

membrane and then treated to remove all contaminating material, leaving just
DNA (Figure 8.9b).

Usually this treatment also results in denaturation of the DNA molecules, so

that the hydrogen bonds between individual strands in the double helix are
broken.

These single-stranded molecules can then be bound tightly to the membrane by

a short period at 80C if a nitrocellulose membrane is being used, or with a
nylon membrane by ultraviolet irradiation.
The molecules become attached to the membrane through their sugarphosphate
backbones, so the bases are free to pair with complementary nucleic acid
molecules

The probe must now be labeled with a radioactive or other type of marker,
denatured by heating, and applied to the membrane in a solution of chemicals
that promote nucleic acid hybridization.

After a period to allow hybridization to take place, the filter is washed to

remove unbound probe, dried, and the label detected in order to identify the
colonies or plaques to which the probe has become bound.

Labeling with a radioactive marker

A DNA molecule is usually labeled by incorporating nucleotides that carry a
radioactive isotope of phosphorus, 32P. Several methods are available:
Nick translation. Most purified samples of DNA contain some nicked
molecules, however carefully the preparation has been carried out, which means
that DNA polymerase I is able to attach to the DNA and catalyze a strand
replacement reaction.

This reaction requires a supply of nucleotides: if one of these is radioactively

labeled, the DNA molecule will itself become labeled.

End filling is a gentler method than nick translation and rarely causes breakage
of the DNA, but unfortunately can only be used to label DNA molecules that
have sticky ends.

The enzyme used is the Klenow fragment, which fills in a sticky end by
synthesizing the complementary strand.

As with nick translation, if the end filling reaction is carried out in the presence
of labeled nucleotides, the DNA becomes labeled.

Random priming results in a probe with higher activity and therefore able to
detect smaller amounts of membrane-bound DNA.

The denatured DNA is mixed with a set of hexameric oligonucleotides of

random sequence. By chance, these random hexamers will contain a few
molecules that will base pair with the probe and prime new DNA synthesis.

The Klenow fragment is used as this enzyme lacks the nuclease activity of DNA
polymerase I and so only fills in the gaps between adjacent primers. Labeled
nucleotides are incorporated into the new DNA that is synthesized.
After hybridization, the location of the bound probe is detected by
autoradiography.
A sheet of X-ray-sensitive photographic film is placed over the membrane. The
radioactive DNA exposes the film, which is developed to reveal the positions of
the colonies or plaques to which the probe has hybridized

Non-radioactive labeling

Radioactive labeling methods are starting to fall out of favor, partly because of
the hazard to the researcher and partly because of the problems associated with
disposal of radioactive waste.
 As an alternative, the hybridization probe can be labeled in a non-radioactive
manner. The use of deoxyuridine triphosphate (dUTP) nucleotides modified by
reaction with biotin, an organic molecule that has a high affinity for a protein
called avidin. After hybridization the positions of the bound biotinylated probe
can be determined by washing with avidin coupled to a fluorescent marker. This
method is as sensitive as radioactive probing and is becoming increasingly
popular.

The same is true for a second procedure for non-radioactive hybridization

probing, in which the probe DNA is complexed with the enzyme horseradish
peroxidase, and is detected through the enzymes ability to degrade luminol
with the emission of chemiluminescence.

The signal can be recorded on normal photographic film in a manner analogous

to autoradiography.

Southern hybridization enables a specific restriction fragment containing agene

to be identified
As well as colony and plaque hybridization analysis, there are also occasions
when it is necessary to use hybridization probing to identify which of a series of
restriction fragments contains a gene of interest.

The first step in using Southern hybridization for this purpose would be to
digest the clone with BamHI and then separate the restriction fragments by
electrophoresis in an agarose gel.

The aim is to use the oligonucleotide probe identify the fragment that contains
the gene. This can be attempted while the restriction fragments are still
contained in the electrophoresis gel, but the results are usually not very good, as
the gel matrix causes a lot of spurious background hybridization
that obscures the specific hybridization signal.

Instead, the DNA bands in the agarose gel are transferred to a nitrocellulose or
nylon membrane, providing a much cleaner environment for the hybridization
experiment.

Transfer of DNA bands from an agarose gel to a membrane makes use of the
technique perfected in 1975 by Professor E.M. Southern and referred to as
Southern transfer.

The membrane is placed on the gel, and buffer allowed to soak through,
carrying the DNA from the gel to the membrane where the DNA is bound.
Sophisticated pieces of apparatus can be purchased to assist this process, but
many molecular biologists prefer a homemade set-up incorporating a lot of
paper towels and considerable balancing skills.

The same method can also be used for the transfer of RNA molecules
(northern transfer) or proteins (western transfer)

Southern transfer results in a membrane that carries a replica of the DNA bands
from the agarose gel.

If the labeled probe is now applied, hybridization occurs and autoradiography

(or the equivalent detection system for a non-radioactive probe) reveals which
restriction fragment contains the cloned gene

Identification methods based on detection of the translation product of the

cloned gene
Hybridization probing is usually the preferred method for identification of a
particular recombinant from a clone library.
The technique is easy to perform and, with modifications introduced in recent
years, can be used to check up to 10,000 recombinants per experiment, allowing
large genomic libraries to be screened in a reasonably short time.

Nevertheless, the requirement for a probe that is at least partly complementary

to the desired gene sometimes makes it impossible to use hybridization in clone
identification.

The main alternative to hybridization probing is immunological screening. The

distinction is that, whereas with hybridization probing the cloned DNA
fragment is itself directly identified, an immunological method detects the
protein coded by the cloned gene.

Immunological techniques therefore presuppose that the cloned gene is being

expressed, so that the protein is being made, and that this protein is not normally
present in the host cells.

Antibodies are required for immunological detection methods

If a purified sample of a protein is injected into the bloodstream of a rabbit, the
immune system of the animal responds by synthesizing antibodies that bind to
and help degrade the foreign molecule.

This is a version of the natural defense mechanism that the animal uses to deal
with invasion by bacteria, viruses, and other infective agents.

Once a rabbit is challenged with a protein, the levels of antibody present in its
bloodstream remain high enough over the next few days for substantial
quantities to be purified.

In the original methods, either the antibody itself was labelled, or the membrane
was subsequently washed with a solution of labelled protein A, a bacterial
protein that specifically binds to the immunoglobulins that antibodies are made
of.

In the more modern methods, the bound antibodythe primary antibodyis

detected by washing the membrane with a labeled secondary antibody, which
binds specifically to the primary antibody.

Several secondary antibody molecules can bind to a single primary antibody

molecule, increasing the amount of signal that is produced and enabling a
clearer detection of each positive colony.
In all three methods, the label can be a radioactive one, in which case the
colonies that bind the label are detected by autoradiography, or nonradioactive
labels resulting in a fluorescent or chemiluminescent signal can be used.
Direct selection (Selection with Antibiotics)
Which means that the cloning experiment is designed in such a way that the
only clones that are obtained are clones of the required gene

To be able to select for a cloned gene it is necessary to plate the transformants

onto an agar medium on which only the desired recombinants, and no others,
can grow.

The only colonies that are obtained will therefore be ones that comprise cells
containing the desired recombinant DNA molecule.

This plasmid carries genes for resistances to four antibiotics: kanamycin,

chloramphenicol, streptomycin, and sulphonamide.

The kanamycin resistance gene lies within one of the 13 EcoRI fragments To
clone this gene, the EcoRI fragments of R6-5 could be inserted into the EcoRI
site of a vector such as pBR322.

The ligated mix will comprise many copies of 13 different recombinant DNA
molecules, one set of which carries the gene for kanamycin resistance
 DNA Fingerprinting
Given the size of the human genome, and our knowledge of genome structure, it
is relatively easy to calculate that each persons genome is unique, the only
exceptions being monozygotic twins (twins derived from a single fertilized
ovum).

This provides the opportunity to use the genome as a unique identifier

DNA profiling (also called DNA testing, DNA typing, or genetic

fingerprinting) is a technique employed by forensic scientists to assist in the
identification of individuals by their respective DNA profiles.

The original technique was called DNA fingerprinting, but with improved
technology the range of tests that can be carried out has increased, and today the
more general term DNA profiling is preferred.

The technique has found many applications in both criminal cases and in
disputes over whether people are related or not (paternity disputes and
immigration
cases are the most common).

The basis of all the techniques is that a sample of DNA from a suspect (or
person in a paternity or immigration dispute) can be matched with that of the
reference sample (from the victim of a crime, or a relative in a civil case).

In scene of- crime investigations, the technique can be limited by the small
amount of DNA available in forensic samples.

Modern techniques use the PCR to amplify and detect minute samples of DNA
from bloodstains, body fluids, skin fragments, or hair roots.

The original DNA fingerprinting technique was devised in 1985 by Alec

Jeffreys, who realised that the work he was doing on sequences within the
myoglobin gene could have wider implications.

The method is based on the fact that there are highly variable regions of the
genome that are specific to each individual.

These are minisatellite regions, which have a variable number of short

repeated-sequence elements known as variable number tandem repeats
(VNTRs,
If molecular geneticists were to clone and sequence the same 250 kb region of
genomic DNA encompassing the cystic fibrosis (CFTR) gene on chromosome 7
from the two homologs of a healthy person, they would find about one
difference in every 1000 bp, or 250 DNA differences in all

When two or more alleles exist at a DNA locus, the locus is considered
polymorphic, and the variations themselves are called DNA polymorphisms.

When a particular polymorphic DNA locus is useful for mapping studies,

disease diagnosis, or any other analytical task, the locus is called a DNA
marker; like a mile marker on a highway, it marks a particular point in the
genome. If that position has no known function, the site is an anonymous locus.

Geneticists Categorize DNA Polymorphisms in Four Different Classes

Single Nucleotide Polymorphisms (SNPs)
The simplest, most prevalent, and most generally useful class of DNA
polymorphisms arises from single base-pair substitutions.

Such base-pair changes, which can be triggered by mutagenic chemicals or

mistakes in DNA replication, are referred to as single nucleotide
polymorphisms, or SNPs.

SNPs account for the vast majority of the total variation

Sequencing the same region of the genome from several individuals can identify
large numbers of SNPs.

Public and commercial research centersand private biotechnology companies

had identified and mapped over 5 million human SNPs.

Although SNPs in coding sequences can alter the aminoacid sequence of a gene
product and have a direct impact on phenotype, the vast majority of SNPs occur
at anonymous loci.

There is no evolutionary advantage or disadvantage to mutations at these

noncoding, nonregulatory loci.

Even though anonymous locus SNPs do not have a direct effect on phenotype,
some lie so close to a disease gene or other genes influencing significant
phenotypic differences (such as positive or negative responses to a particular
medication) that they can serve as DNA markers: specific DNA loci with
identifiable variations.

Medical researchers can use such markers to identify and follow phenotypic
differences in groups of people.

Microsatellites
The genomes of humans and other complex organisms are loaded with loci
defined by repeated sequences.

The repeating unit can be as short as a single base pair or as long as tens of
kilobases. The number of repeats can vary between two and thousands.

microsatellites, which are DNA elements composed of one-, two-, or three-

base sequences repeated in tandem 15100 times. Microsatellites are also called
SSRs (simple sequence repeats)

Examples are AAAAAAAAAAAAAAA or

CACACACACACACACACACACA. In the mammalian genome, the CA-
repeat microsatellite occurs on average once
in every 30,000 bp.

Minisatellites

Minisatellites, a second important category of genomic DNA repeats, are larger

than microsatellites.

The repeating units that compose them are 20100 bp long, and each unit is
repeated up to thousands of times per locus. This gives each minisatellite locus
a total length of 0.520 kb

Like microsatellites, minisatellites arise from random events and are dispersed
throughout the genomes of all vertebrates.

Using several unrelated minisatellite sequences as hybridization probes can

provide an overview of the whole
genome.

The frequency of such multilocus minisatellites is about 1 per 100,000 bp, for a
total of in the whole human genome.

Deletions, Duplications, and Insertions at Nonrepeat Loci (Indels)

The DNA changes in this broad category are the result of mutagenic events that
expand or contract the length of a nonrepetitive DNA locus (which excludes
micro- and minisatellites) by deleting, duplicating, or inserting one or more base
pairs.

These mutations, commonly called indels, can range in size from a one or few
base pairs to multiple megabases.

Detecting DNA Genotypes of Different Types of Polymorphisms

One general approach to determining an individuals genotype at a particular

polymorphic locus is to extract genomic DNA from the individual, use PCR to
amplify the locus, and then sequence the PCR product.

RFLP- Restriction Length Polymorphism

DNA fingerprinting relies on the unique pattern made by a series of DNA

fragments after separating them according to length by gel electrophoresis.

DNA samples from different suspects, the victim, and samples from the crime
scene are first purified. Restriction enzymes cut the DNA samples into
fragments of different lengths.

Consequently, the variation in the size of the fragments and hence of their
positions on an agarose gel is due to differences in where cutting occurs. Thus
differences in patterns between individuals are due to differences
in the base sequence of their DNA.

The nucleotide differences that cause the fragment lengths to vary are called
restriction fragment length polymorphisms.
There is believed to be approximately one difference in every 1000 nucleotides
between nonrelated individuals.
The steps involved in DNA fingerprinting are as follows
1. The DNA is cut with a restriction enzyme.

2. The DNA fragments are separated according their length or molecular weight
by gel electrophoresis.

3. The fragments are visualized by Southern blotting. After transfer of the

separated fragments from the gel to nylon paper a radioactively labeled DNA
probe is added. The probe will bind to those DNA fragments whose DNA
sequences are complementary to the probe.
4. An autoradiograph is made by covering the blot with radiation-sensitive film.

This will show the location of those DNA fragments that reacted with the
radioactive probe.

There are many different restriction enzymes, most with unique cutting
properties.

In practice several different enzymes are used with the same DNA samples,
giving different sets of fragments for different people. These can be compared
with DNA samples taken from a victim or found at a crime scene.

Because there is so much genetic diversity, RFLP patterns from different people
can vary a lot. Even if mutations have changed a small percentage of the target
sequence around the cut site, there will usually still be enough similarity for
binding of the probe to occur.

The entire process requires several weeks to finish.

The final product of a DNA fingerprint is an autoradiograph that contains at

least five essential lanes.

The markers are standardized DNA fragments of known size, which have been
radioactively labeled. These help determine the size of the various fragments.

The control is DNA from a source known to react positively and reliably to
the DNA probes and shows whether the test has worked as expected. The
experimental lanes have samples from the victim, the defendant, and the crime
scene. In this example, blood from the defendants clothing was compared with
his/her own blood and the victims blood.

The DNA from the clothing actually matches that of the victim. Two variants of
DNA fingerprinting have been usedsingle-locus probing (SLP) and multiple-
locus probing (MLP). In SLP, a probe is used that is specific for a single site,
that is, a single locus, in the genomic DNA. Because humans are diploid, an
SLP probe will therefore normally give rise to two bands from each person for
each particular locus.

This assumes that the chosen locus shows substantial allelic variation.
Occasional persons will be homozygous and hence show only a single band.
For full identification using SLPs, it is necessary to run several reactions, each
using a different SLP probe. SLP analyses use smaller amounts of material than
MLP and are easier to interpret and compare.

Statistical analysis and population frequencies are possible using SLP data.

Randomly amplified polymorphic DNA

Randomly amplified polymorphic DNA (RAPD), allows the researcher to

compare the genetic relatedness of two DNA samples .
First, two DNA samples are isolated from two different organisms. Thus there
are two samples of target DNA, which are compared by using the same set of
primers.

As before, the sequence information is unknown, but rather than using

degenerate primers, primers with a randomly chosen sequence are made.

If the target DNA were a sample from a large genome, such a primer would
bind far too many times. In practice longer primers of around 10 bases are often
suitable.

The random primer is mixed with nucleotides, Taq polymerase, and each of the
target DNA samples as for normal PCR reactions.

In order to amplify any target DNA fragments, two of the random primers must
bind to the target DNA, on opposite strands, usually within a few thousand
bases.

The results of the two PCR reactions are compared using gel electrophoresis.
The number and size of PCR products will vary for the two samples.

If two organisms are very closely related, then their DNA will be close in
sequence. Hence, the PCR products will be very similar with only one or two
different fragments.

If the pattern of PCR products is totally different, then the two organisms are
not related. Comparing RAPDs from two organisms can thus give an estimate
of relatedness.
UNIT IV APPLICATIONS r DNA TECHNOLOGY

Transgenic Plants
Genetic modification of plants probably began through selection of novel types
about 10 000 years ago when human agricultural activities were initiated

Traditional plant breeding methods have been very successful, providing the
volume of food required to allow the world population to grow to its present
scale

Traditional methods alone will not be able to keep pace with the growing
demands for food, fibre and fuel.

Much of the early increase in grain production resulted from an increase in an

area under cultivation, irrigation, better agronomic practices and improved
cultivars.
It may take 10 or more years to transfer a trait from a donor species into a crop
cultivar via conventional strategies.

From the genetic point of view, one single cross between two plants used in
conventional breeding puts two sets of about 15 00025 000 genes together,
i.e. a genetic modification at a massive level

In contrast, by means of modern biotechnological methods only few genes are

modified leaving the rest of the genome unaltered.

Plant biotechnology offers breeders access to an infinitely wide array of novel

genes and traits, which can be inserted through a single event into high-yielding
and locally adapted cultivars.

Although the plant transformation technology has been developed in 1983, the
first genetically transformed crop reached into markets during the mid-1990s.

In 2007, the global area of biotech crops increased for the twelfth consecutive
year
at an annual growth rate of 12%, with a total area of 114.3 million hectares in
23 countries

Transgenic crops have contributed more than US$ 23 billion to the economies
of
developing as well as developed countries (nearly 90% of the transgenic crops
are planted by resource-poor farmers)

The main approaches used to produce improved transgenic plants with

commercial or agricultural applications are mentioned in the following sub-
headings.

1. Improvement of crop productivity

2.Improvement of crop nutritional qualities
3.Genetic modifications of plants to generate useful Products

1. Improvement of crop Productivity

Abiotic stress
Abiotic stress is defined as the negative impact of non-living factors on the
living organisms in a specific environment
Abiotic stress is the most harmful factor concerning the growth and productivity
of crops worldwide

Abiotic stress comes in many forms. The most common of the stressors are
high temperatures (heat), cold, drought, flood, and edaphic conditions like
salinity of soil

Trnsgenic plants for Heat tolerance

Most crops are affected by daily/seasonal fluctuations in high day and/or night
temperatures.

Heat stress is often defined as the rise in temperature beyond a Threshold level
for a period of time sufficient to cause irreversible damage to plant growth and
development.

In general, a transient elevation in temperature, usually 1015 C above

ambient, is
considered heat shock or heat stress.

Heat tolerance is generally defined as the ability of the plant to grow and
produce economic yield under high temperatures.

Heat stress due to high ambient temperatures is a serious threat to crop

production worldwide

Gaseous emissions due to human activities are substantially adding to the

existing concentrations of greenhouse gases,

At very high temperatures, severe cellular injury and even cell death may occur
within minutes, which could be attributed to a catastrophic collapse of cellular
organization

Direct injuries due to high temperatures include protein denaturation and

aggregation, and increased fluidity of membrane lipids. Indirect or slower heat
injuries include inactivation of enzymes in chloroplast and mitochondria,
inhibition of protein synthesis, protein degradation and loss of membrane
integrity
These injuries eventually lead to starvation, inhibition of growth, reduced ion
flux, production of toxic compounds and reactive oxygen species (ROS)

Immediately after exposure to high temperatures and perception of signals,

changes occur at the molecular level altering the expression of genes and
accumulation of transcripts, thereby leading to the synthesis of stress-related
proteins as a stress tolerance strategy.

Expression of heat shock proteins (HSPs) is known to be an important adaptive

strategy in this regard

The tolerance conferred by HSPs results in improved physiological phenomena

such as photosynthesis, assimilate partitioning, water and nutrient use
efficiency, and membrane stability. Such improvements make plant growth and
development possible under heat stress.

Genetic engineering have provided additional tools, which could be employed

to develop crops with improved heat tolerance and to combat this universal
environmental adversary

several genes responsible for inducing the synthesis of HSPs have been
identified
and isolated in various plant species, including tomato and maize

Initial research on molecular manipulation to improve plant heat tolerance

focused on production of enzymes that detoxify reactive oxygen species,
including SOD.

Reactive oxygen species are induced by most types of stresses and their
production has been envisaged in stress cross-tolerance. In addition to
increased production of Super Oxide Desmutase (SOD), many other potential
approaches can be utilized to detoxify Reactive Oxygen species (ROS)

Development of plants capable of higher production of glycinebetaine through

transformation with the BADH gene has been suggested as a potentially
effective method to enhance heat tolerance in plants

Transgenic over expression of certain HSFs and HSF-fusion proteins results in

an expression of HSPs at normal temperature

MT-sHSP gene exhibits thermotolerance in transformed tobacco with the

tomato MT-sHSP gene
Drought tolerance

Adverse environmental factors, of which water scarcity represents the most

severe constraint to agriculture, account for about 70 percent of potential yield
loses worldwide

Global warming is also predicted to affect most severely developing countries,

where agricultural systems are most vulnerable to climatic conditions and where
small increases in temperature are very detrimental to productivity.

It is predicted that 600,000 square km currently classed as moderately

constrained water scarcity will become severely limited for water source

Water becomes an increasingly scarce and precious commodity. It is thus

essential to improve water use efficiency in agriculture.

The development of crop varieties with increased tolerance to drought, both by

conventional breeding methods and by genetic engineering, is also an important
strategy to meet global food demands with less water

Although conventional breeding for drought tolerance has and continues to have
some success, it is a slow process that is limited by the availability of suitable
genes for breeding.

The development of tolerant crops by genetic engineering, on the other hand,

requires the identification of key genetic determinants underlying stress
tolerance in plants, and introducing these genes into crops.

Drought triggers a wide array of physiological responses in plants, and affects

the activity of a large number of genes: gene expression experiments have
identified several hundred genes which are either induced or repressed during
drought

Therefore, the genetic control of tolerance to abiotic stresses is not only very
complex, but is also highly influenced by other environmental factors and by
the developmental stage of the plant.
Plant cells are required to maintain water balance. To maintain this water
balance, plants absorb water when water potential is negative Cells can
decrease their water potential through the accumulation of solutes, such as
sugars, amino acids, organic acids and ions especially potassium (K+).
As cellular enzymes are severely inhibited by the presence of ions, these must
be removed from the cytosol (the ground fluid substance of the cell) and stored
in special storage cell organelles, the vacuoles. Compatible solutes that
accumulate in the cytosol and do not interfere with enzymatic reactions
comprise sugar, alcohols (mannitol and sorbitol), the amino acid proline, and
glycine betaine. The synthesis of these compounds by the plant enhances
tolerance to drought.
The plants response to drought is accompanied by the activation of genes
involved in the perception of drought stress and in the transmission of the stress
signal.
One group of genes that encode proteins that protect the cells from the effects
of desiccation. These genes include those that govern the: accumulation of
compatible solutes; passive transport across membranes; energy-requiring water
transport systems; and protection and stabilization of cell structures from
desiccation and damage by reactive oxygen species
A second group of genes activated by drought is comprised by regulatory
proteins that further regulate the transduction of the stress signal and modulate
gene expression

Two of the pathways are dependent on the hormone ABA, and two are ABA-
independent. These pathways are also implicated in the perception and response
to additional stress factors, including cold, high temperature and salinity

Many of the genes known to be involved in stress tolerance have been isolated
initially in Arabidopsis. The introduction of several stress-inducible genes into
plants by genetic engineering has resulted to increased tolerance of transgenics
to drought, cold and salinity stresses

ABA levels in the plant greatly increase in response to water stress, resulting in
the closure of stomata thereby reducing the level of water loss through
transpiration from leaves and activate stress response genes. The reaction is
reversible: once water becomes available again, the level of ABA drops, and
stomata re-opens

The transcription factors DREB1 and DREB2, are important in the ABA-
independent drought tolerant pathways, that induce the expression of stress
response genes. Over-expression of the native form of DREB1, and of a
constitutively active form of DREB2, increases the tolerance of
transgenic Arabidopsis plants to drought, high salinity and cold.
Proline is the most widely distributed osmolyte; it occurs in plant and in many
other organisms. Its accumulation correlates with tolerance to drought and salt
stress
Roles: osmotic adjustment, membranes protection, sink of energy and
reducing power

Herbicide resistance/ tolerance

Weed control is one of the farmers biggest challenges.

Poorly controlled weed drastically reduce crop yield and quality.

Conventional strategy relies on chemicals.

Excess use of these chemicals in the past two decades has led to the
accumulation of herbicide in the soil causing severe pollution.

The herbicide resistance transgenic crops resolves many of the problems

associated with herbicide non selectivity and also facilitate the low or no tillage.

Herbicide resistant transgenic crops are the most accepted among the transgenic
crops.
In 2010 herbicide resistance deployed in soybean, maize , canola, cotton,
sugarbeat and alfalfa occupied 61% of transgenic crop and planted in 89.3
million hectares.

North America and USA are the leading countries in herbicide resistant crop
growing.

Strategies for herbicide resistance

Herbicides act by inhibiting the protein or enzyme involved in the vital
metabolic pathways of plants.

Aminoacid biosynthesis, nitrogen metabolism and photosynthesis are the

important target pathways for different herbicides.

Therefore the herbidide resistant transgenic production relies on the mechanism

of action of herbicides.

Detoxification mechanism:
Detoxification of herbicide by introducing gene encoding for enzyme which
cleaves the herbicides in non toxic compounds.

A detoxification gene bar isolated from Streptomyces hygroscopicus was found

useful to detoxify the herbicide phosphinothricin (a compunnd blocks the
glutamine synthesis) which is the active ingredient of commercial herbicide
Basta.

This gene produces the enzyme phosphinothricin acetyl transferase which

detoxify the phosphinothricin.

Transgenics containing bar gene was developed in tobacco, wheat maize,

sugarcane canola, soybean and rice.

Similarly a gene (bxn) gene isolated from Klebsiella ozonae encoded for
nitrilase detoxifies the herbicide bromoxynil which inhibits the photosynthetic
system. This gne is transferred to tobacco and conferred the resistance against
the herbicide bromoxynil

Development of transgenics insensensitive to herbicides

This approach involves development of transgenic crop resistant to herbicide

Glyphosate ( RoundUP trade name)

Glyphosate is a broad spectrum herbicide that inhibits the enzyme 5Enol

PyruvylShikimate 3 Phosphate synthase (EPSPS) involved in the aromatic
amino acid biosynthesis.
The bacterial gene aroA isolated from Agrobacterium which encodes for EPSPS
enzyme insensitive to glyphosate is developed in cotton and canola.

This mechanism gives tolerance to herbicides.

Introduction of gene ALS encoding for the herbicide insensitive acetolactate

synthetase enzyme from E.Coli. Sulfonyl urea blocks the enzyme ALS and
affects the branched chain amino acid synthesis.

Over expressing mechanism

The third approach is over expression of the gene encoding for enzyme which
is the herbicide target in plants.
Over expression of the enzyme in the crops provide resistance to herbicide by
providing large number of enzyme molecules. The herbicides blocks only part
of the enzyme molecules. Hence the enzyme is available for synthesis.

Over expression of EPSPS enzyme was achieved in soybean

Salt tolerance transgenic plants

Agricultural productivity is severely affected by soil salinity because salt levels

that are harmful to plant growth affect large terrestrial areas of the world.

The damaging effects of salt accumulation in agricultural soils have influenced

ancient and modern civilizations.

It is estimated that 20% of the irrigated land in the world is presently affected
by
salinity.

The development and use of crops that can tolerate the high levels of salinity in
the soils would be a practical contribution towards addressing the problem.

Physiologically, salinity (i) imposes an initial water-deficit that results from the
relatively high solute concentrations in the soil, (ii) causes ion-specific stresses
resulting from altered K+/Na+ ratios and (iii) leads to build up in Na+and Cl-
concentrations that are detrimental to plants.

Plants respond to salinity using two different types of responses. Salt-sensitive

plants restrict the uptake of salt and adjust their osmotic pressure by the
synthesis of compatible solutes (e.g. proline, glycinebetaine and sugars)

Salt-tolerant plants sequester and accumulate salt into the cell vacuoles,
controlling the salt concentrations in the cytosol and maintaining a high
cytosolic K+/Na+ ratio in their cells.
(i) extrusion of NaC ions out of the cell and (ii) vacuolar compartmentation of
Na+ ions.

AtHKT1 gene ( isolated from Arabidopsis) played a role in long-distance Na+

transport and Na+ circulation in the plant, with AtHKT1 mediating Na+ loading
into the leaf phloem and NaC unloading from the root phloem sap

AtSOS1 from Arabidopsis thaliana has been shown to encode a plasma

membrane Na+ /H+ antiporter

The over expression of SOS1 improved the salt tolerance of Arabidopsis,

demonstrating that improved salt tolerance can be attained by limiting Na+
accumulation in plant cells

Similar results were obtained when the plasma membrane Na+/HC antiporters
SOD2 from Schizosaccharomyces pombe and nhaA from Escherichia coli were
over expressed in Arabidopsis and rice, respectively.

The compartmentation of Na+ ions into vacuoles also provides an efficient

mechanism to avert the toxic effects of Na+ in the cytosol.

The over expression of AtNHX1, a vacuolar Na+/HC antiporter, in Arabidopsis

resulted in transgenic plants that were able to grow in high concentrations of
salt

Over expression f AtNHX1 gene in tomato and canola withstand up to 200mM

sodium chloride.

The AtNH1 gene has been transferred to wheat and maize showed increased
tolerance.

The over expression of the rice vacuolar Na+/H+ antiporter (OsNHX1) in rice
also conferred salt tolerance to the transgenic plants

Although there have been many successes in developing stress-tolerant

transgenics in model plants such as tobacco, Arabidopsis or rice, there is an
urgent need to test these successes in other crops.
Disease resistance

Viral resistance

Coat protein Mediated resistance

There are 40 type of virus affecting crop plants Trangenic plants with viroids
(coat protein genes) was found with reduced symptoms when challenged with
the same virus. The plants were transferred with part of the virus genome- also
called cross protection

CP-mediated resistance is based on prevention of the uncoating of the

challenge virus as it enters the plant cell, which interferes with the
translation and replication processes

CP-mediated resistance in transgenic plants depends on the expression

level of the transgene CP, and a higher level of transgene expression
elicits better protection.

CPMR can provide either broad or narrow protection; for example, the CP of
TMV provided effective levels of resistance to closely related strains of TMV
and decreasing levels of resistance to tobamoviruses that share less CP sequence
similarity

However the CP gene of papaya ringspot virus (PRV) strain HA provided

resistance in papaya only to strain HA

The transgenic Tobacco with Tobacco Mosaic virus protein were resistant to
TMV virus.

Tobacco Streak Virus (TSV) resistance in tobacco is also produced . This cross
protection was extended to Solanaceae crops
Potato- Potato virus X (PVX) and Potato virus Y (PVY) , Tomato TMV

Cucurbitaceae crop Squash- Zucchini Yellow Mosaic virus(ZYMV) and

Watermelon Mosaic virus (WMV)

it is clear that knowledge of the three-dimensional structures of other CP

molecules and the role of CP in regulating infection and/or replication will
significantly aid the design of mutant CPs that have increased efficacy and
breadth of protection.
Replicase-mediated resistance

Genes that encode complete or partial replicase proteins can confer near
immunity to infection that is generally, but not always, limited to the virus
strain from which the gene sequence was obtained.

Replicase-mediated resistance (Rep-MR) to TMV was first described in

transgenic plants that contain a sequence encoding a 54 kDa fragment of
replicase, although the protein fragment was not detected.

It was suggested that certain examples of Rep-MR are RNA- rather than
protein-mediated. The exact mechanisms that are involved in Rep-MR are not
known,

A truncated mutant of replicase derived from a cucumber mosaic virus (ChlV)

subgroup I virus conferred high levels of resistance in tobacco plants to all
subgroup I ChlV strains,

TMV replicase gene transgenics encodes putative component of replicase

complex resistant to TMV infection

Movement proteins
Movement proteins (MPs) are encoded by plant viruses and enable infections to
spread between adjacent cells (local spread) as well as systemically.

Intercellular spread involves plasmodesmata, the channels that traverse plant

cell walls and provide symplastic continuity between cells and tissues.

The short distant cell to cell movement of plant viruses is most probably
trafficked through the plasmodesmata

TMV movement protein transferred to tobacco plants were resistant to TMV.

Transgenic plants that contain dMP from TMV show resistance to several
Tobamoviruses as well as to cauliflower mosaic virus (CaMV) and other viruses

Antisense RNA

Mirror sequence of mRNA sequence is transcribed antisence coat protein

genes of Cauliflower Mossaic virus (CAMV) stopped the coat protein
production

TMV virus resistance in Tobacco exhibited viral tolerance.

Resistance against fungal pathogens
The majority of phytopathogenic fungi belong to the Ascomycetes and
the Basidiomycetes.

The fungal pathogens caused more than 50% yield loss in crop plants

The fungal diseases were successfully controlled by fungicides but it caused

cevere damage to environment.

Antifungal compounds include antifungal proteins from

Plants and lower organisms and metabolites like phytoalexins are useful to
control Fungal disease

genes encoding many antifungal proteins which can inhibit fungal growth in
vitro have been exploited to make fungus-resistant transgenic plants

Some of these proteins are: Pathogenesis-related proteins, Ribosome-

inactivating proteins, Small cystein-rich proteins, Lipid transfer proteins,
Storage albumins, Polygalacturonase inhibitor proteins (PGIPS),

Antiviral proteins, and Non-plant antifungal proteins.

Pathogenesis-related proteins:

PR proteins are induced during hypersensitive response (HR) and also during
systemic acquired resistance (SAR) and therefore are thought to have a role in
natural defense or resistance of plants against pathogens

PR proteins have been grouped into five families based on primary structure,
serological relatedness and enzymatic and biological activities.

PR2 and PR3 type proteins are the fungal cell wall hydrolysing enzymes,
glucanase and chitinase respectively.

These proteins can inhibit the fungal growth in vitro by causing lysis of hyphal
tips

PR5 proteins (thaumatin-like or AP24 or osmotin), in all probability, cause lysis

of the pathogen by permeabilizing the fungal cell wall

The first report on developing fungus-resistant transgenics came in 1991.

Broglie et al. constitutively expressed bean chitinase gene in tobacco and
Brassica napus and the plants showed enhanced resistance to Rhizoctonia
solani.
Since then there have been a number of reports on transgenics developed by
constitutively expressing PR-protein genes in Canola, carrot, Grapewine, Rice,
potato, Tobacco, Tomato, sugarcane, barley and wheat.

Plant ribosome-inactivating proteins:

Plant Ribosome inactivating proteins (RIPs) have N-glycosidase activity and

they remove an adenine residue from 28S rRNA. As a consequence, the 60S
ribosomal subunit is not able to bind to elongation factor 2, resulting in
inhibition of protein elongation.

Tobacco plants constitutively expressing a RIP encoding DNA sequence of

barley showed better resistance to R. solani

Small cystein-rich proteins:

In addition to PR proteins, there are other plant proteins which have antifungal
activities. A number of small cystein-rich proteins form a separate group of
antifungal polypeptides.

Some of these are chitin-binding proteins, plant defensins and thionins.

Transgenic tomato plants expressing hevein gene showed fewer symptoms on

slices of transgenic tomato fruits compared to controls when infected with
Trichoderma hamatum

One of the best studied plant defensins is Rs-AFP2 (Raphanus sativus

antifungal protein-2). Transgenic tobacco plants producing RS-AFP2 show
enhanced resistance to the foliar pathogen Alternaria longipes

Lipid transfer proteins:

The proteins are so named because of their ability to stimulate the transfer of a
broad range of lipids through the membrane in vitro and might be involved in
secretion of or deposition of extracellular lipophillic materials such as cutin or
wax.

The same group developed transgenics in tobacco and Arabidopsis with

constitutively expressing barley LTP2 protein and reported enhanced tolerance
to Pseudomonas syringae

Phytoalexins
Phytoalexins are antimicrobial low molecular weight secondary metabolites
produced in plants following pathogen attack and are believed to have a role in
plant defense

The expression of stilbene synthase (or resveratrol synthase) gene resulted in

the production of resveratrol, a stilbene-type phytoalexin. Such transgenics
showed enhanced resistance to B. cinerea.

Similar transgenic plants were developed in rice, tomato, barley and wheat and
were shown to have increased resistance to Magnaporthe grisea, P. infestans
and B. cinerea respectively

Resistance genes from plants

All plants have passive defense lines such as cell walls, wax layers and
chemical barriers against pathogens. If the pathogen overcomes this first line of
defense, there is a second line of defense, which is mounted by proteins
encoded by specific resistance (R) genes.

This line of defense is best described genetically by the gene for gene model. It
requires a pathogen protein encoded by an avirulence (Avr) gene to be
recognized by a plant protein encoded by a resistance (R) gene. This activates
an array of defense mechanisms, including the hypersensitive response.

R genes transferred to Tomato, tobacco, rice and barley showed better tolerance
against fungal pathogens.

Broad spectrum disease resistance using SAR

One of the effective strategies for broad spectrum plant disease resistance has
been to exploit SAR (Systemic Acquired Resistance) pathway.

Over expression of Prf gene induces SAR (necrosis with hypersensitivity) in

tomato in a pathogen independent manner.

The induction of SAR without deleterious side effects makes Prf-mediated

transgenic SAR a target for production of broad spectrum-enhanced resistance
in agricultural crops

Bacterial Disease resistant transgenics

Although bacteria causing cevere damage to crop species little progress has
been made in bacterial resistant transgenics.
Lysozymes are a class of antibacterial proteins found in many plants.

They cleaves the glycosydic bonds of bacterial cell wall.

The bacteriophage T4 lysozyme is very effective enzyme for lysis of a wide

range of bacteria

Erwinia carotovora is apotential pathogen of potato can be controlled in

transgenic potato engineered with T4 lysozyme gene secretes in plant
intercellular spaces.

Transgenic tobacco expression the fusion protein fromT4 Lysozyme and alpha
amylase expressed resistance against bacterial pathogen.

Transgenic tobacco plant procucing Cercopin (family of small strong basic

protein) analogue which confers reistance against bacterial wilt disease.

Thionins are a family of 5 KD cystein rich protein in Cereal endosperm.

Barley pants transferred with thionins had enhance the reistance against
Pseudomonas pathogen.

(Erwinia -soft rot, Pseudomonas-Blight)

Nematode resistance

Nematodes damages the crop plant seriously.

Root-knot nematodes (Melodiogyne. Sp.) Cause a loss of 100billion dollars.

Cyst nematodes (Heterodera spp.) are also causing considerable damage

Chemical protection like Methyl bromide is costlier and damage environment.

The genes for inhibition of proteinase (cystatin) reduces the protein digestion
capability in tobacco and conferred resistance against Cyst nematodes

A strong promoter with these genes are delivered through Agrobacterium

rhizogenes which promotes hairy root and expression of these proteinase
inhibitors limited the growth of nematodes

This mechanism was successful in tomato plants.

Insect resistant plants

Losses due to pests and diseases have been estimated at 37% of the agricultural
production world-wide, with 13% due to insects

Different approaches to obtain insect-resistant plants are presently being

explored. The main approach uses d-endotoxin coding sequences originating
from the bacterium Bacillus thuringiensis and the culmination of this process
occurred in 1996 when the first generation of insecticidal plants were
introduced on the market.

Other strategies use plant derived genes, such as those encoding enzyme
inhibitors or lectins.

The B. thuringiensis toxins

The bacterium B. thuringiensis was first discovered in Japan in 1902 in a

silkworm rearing unit.
In 1911, it was again isolated in a flour moth population and characterized by
Berliner in Thuringen (Germany). B. thuringiensis is a gram positive bacterium
that synthetizes insecticidal cristalline inclusions during sporulation.

The crystalline structure of the inclusion is made up of protoxin subunits, called

d-endotoxins.

Most B. thuringiensis strains produce several crystalline proteins (Cry proteins),

each of which shows a rather narrow host range

At least 90 genes encoding protoxins from a wide range of B. thuringiensis

isolates have been isolated and sequenced.
The genes were first classified in different classes cryI, cryII, cryIII based on
protein structural homologies and host range.
CryI toxins are active against Lepidoptera while CryIII are active against
Coleoptera.
More recent analysis of the nucleotide sequence reveals that this classification is
not necessarily based on homology or evolutionary relationships, and a new
nomenclature has been proposed

The d-endotoxins are solubilized in the insect midgut and are activated by gut
proteases that cleave the protein into a smaller polypeptide, the toxin.
This toxin binds to the surface of epithelial cells in the midgut, inducing lesions
that destroy the cells and lead to the death of the insect.
B. thuringiensis was first used as a bioinsecticide and the main advantage of
such formulations is that they are harmless to humans, mammals and to the non-
target fauna.
Of the bioinsecticides in use, 90% are based on B. thuringiensis, representing
in 1992 2% of the global world pesticide market.

The approach consisting in the transfer and expression of B. thuringiensis toxin-

encoding genes into plants has attracted much attention.
Indeed, such a system allows the entire plant to be protected, especially against
insects such as borers that infect plant parts that sprays often cannot reach.

Furthermore, the toxin affects the more susceptible early instar stages of the
insect and the system is environmentally safe because the product is retained
within the plant tissues.

The first results concerning the transfer of B.thuringiensis genes in tobacco and
tomato were published in 1987.
Since then, B. thuringiensis genes have been transferred to a number of other
crop species such as cotton, rice, maize..., with Lepidoptera as the main targets.

In most cases, the B. thuringiensis toxin coding sequences have been placed
under the control of constitutive promoters (CaMV 35S promoter

Bt genes and cotton

Cotton the most important commercial crop of India, often referred as the White
Gold, consumes more than 45% of the total pesticides used in our country. The
most important insect pests that affect cotton production are jassids, white fly,
aphids and thrips among the sap sucking pests and boll worms (American, Pink
and Spotted) and Spodoptera among the leaf eating caterpillars.

Of these cotton pests, the American boll worms alone cause yield reduction
upto 40 70 % under severe incidence

The Bt transgenic cotton (Bollgard of Monsanto) has thus been developed

successfully in USA, which has the ability to control the bollworms during crop
growth effectively.
Ever since three Bt cotton hybrids have been approved for commercial
cultivation in India during 2002, there was a sharp increase in area under
cultivation of such hybrids from a mere 72,000 ac in 2002-03 to 30,00,000 ha in
2006-07. So far, 59 Bt hybrids have been approved in different cotton growing
zones by GEAC (Genetic Engineering Approval Committee, Government of
India)

Bt cotton hybrids exhibited excellent control of American Boll worm and

reduced the use of insecticides leading to create eco-friendly environment
without compromising on profitable yield.

The genetic resistance, one of the important pest management strategy, is

available in cotton gene pool against the sap sucking pests such as jassids,
whitefly etc and using this several resistant / tolerant varieties and hybrids have
been developed and released in India.

cloning and transferring the genes encoding the toxic crystal - endo toxin
protein from the soil bacterium Bacillus thuringiensis. The Bt transgenic cotton
(Bollgard of Monsanto) has thus been developed successfully in USA, which
has the ability to control the bollworms at the early stages of crop growth (upto
90 days) effectively

The first commercial Bt cotton variety was released in USA by M/S. Monsanto
(Bollgard), which contains Cry 1Ac gene of Bacillus thuringiensis. Bt cotton is
commercially grown in several countries like China, Australia, Mexico, South
Africa, Argentina, India, Indonesia etc. World wide the area under Bt cotton
keep increasing year by year. Overall, about 12% of the world cotton is now
planted with Genetically Modified varieties / hybrids (GMO)

As compared to insecticide control of bollworms, Bt cotton technology will not

harm non-target beneficial insects, reduction in production cost, increased
profit, reduced farming risk and improved economic outlook for cotton.

Use of plant-derived genes

The synthesis of antimetabolic proteins, which interfere with the digestive
processes in insects, is a defence strategy that plants use extensively.
Such proteins can be synthetized constitutively, in tissues that are particularly
vulnerable to attacks such as seeds, or can be induced by mechanical wounding,
as it is the case when chewing insects feed on leaves.

Proteinase inhibitors
proteinase inhibitors (PIs) can be divided in four classes, inhibiting serine,
cysteine, metallo- or aspartyl proteases.

Plant PIs are small proteins and most of the serine PIs possess two active sites
which inhibit trypsin and chymotrypsin. Serine and cysteine

PIs are abundant in seeds and storage tissues of plants

The first gene to be successfully transferred to another plant species resulting in

enhanced insect resistance was isolated from cowpea and encoded the trypsin:
trypsin inhibitor CpTI

The CpTI gene has been transferred to Tobacco Rice, potato and this protein is
an effective antimetabolite against a range of field and storage pests

Lepidoptera and Diptera possess mainly serine proteinases and the obtention of
plants more resistant to Lepidoptera through the use of serine PIs of different
origins has been reported

a-Amylase inhibitors
The commun bean, Phaseolus 6ulgaris, contains a family of related seed
proteins (PHA-E and -L, arcelin, and a-amylase (a-AI)).
PHA-E and -L are classical lectins with strong agglutinin activity,

The introduction and expression of the bean a-AI gene under the control of the
5% and 3% regions of the bean phytohemagglutinin gene in pea confers
resistance to the bruchid beetles,

The transfer of this a-AI gene to Azuki bean confered resistance to three species
of
bruchids

Lectins
Lectins are carbohydrate-binding proteins found in many plant tissues, and are
abundant in the seeds and storage tissues of some plant species.
The toxicity of this type of protein to mammals and birds is well documented.
The toxicity of different lectins towards insects has been observed

Lectins such as those purified from snowdrop or garlic are toxic to insects but
not to mammals.
Tobacco plants expressing a pea lectin were shown to be toxic to the
Lepidoptera Heliothis 6irescens and potato plants expressing the snowdrop
lectin (GNA) were toxic to the Lepidoptera Lacanobia

Chitinases, tryptophan decarboxylase

Insects contain chitin, not only as an exoskeletal material, but also at the level of
the peritrophic membrane.
Chitinase activity could therefore interfere with digestion.
The expression of a bean chitinase in potato causes no deleterious effect to
a Lepidoptera, the expression of a chitinase of insect origin in transgenic
plants seemed to be more effective in causing larval mortality to a beetle,
Oryzaephilis mercator

The production of tryptamine in transgenic tobacco causes a decrease

in whitefly (Bemisia tabaci) pupae emergence

Bean chitinase gene has been transferred to potato and delivered resistance
against Lacamobia oleracea

2. Improvement of crop nutritional qualities

Genetic modification of food crops offers the possibility of enhancing the

nutritional content of the food

1.The development of Golden Rice, genetically engineered to produce -

carotene (pro-vitamin A) in the seeds

Golden rice is a variety of Oryza sativa rice produced through genetic

engineering to biosynthesize beta-carotene, a precursor of pro-vitamin A in the
edible parts of rice. The scientific details of the rice were first published
in Science in 2000. Golden rice was developed as a fortified food to be used in
areas where there is a shortage of dietary vitamin A.
 In 2005 a new variety called Golden Rice 2 was announced which produces up
to 23 times more beta-carotene than the original variety of golden rice. Neither
variety is currently available for human consumption.

Golden rice was created by Ingo Potrykus of the Institute of Plant Sciences at
the Swiss Federal Institute of Technology, working with Peter Beyer of
the University of Freiburg. The project started in 1992, and at the time of
publication in 2000, golden rice was considered a significant breakthrough in
biotechnology, as the researchers had engineered an entire biosynthetic
pathway.
Golden rice was designed to produce beta-carotene, a precursor of vitamin A, in
the part of rice that people eat, the endosperm. The rice plant can naturally
produce beta-carotene, which is a carotenoid pigment that occurs in the leaves
and is involved in photosynthesis. However, the plant does not normally
produce the pigment in the endosperm, since photosynthesis does not occur in
the endosperm.
Golden rice was created by transforming rice with two beta-carotene
biosynthesis genes:

1. psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus)

2. crtl from the soil bacterium Erwinia uredovora
(The insertion of a lyc (lycopene cyclase) gene was thought to be needed, but
further research showed it is already being produced in wild-type rice
endosperm.)
The psy and crt1 genes were transformed into the rice nuclear genome and
placed under the control of an endosperm-specific promoter, so they are
only expressed in the endosperm. The exogenous lyc gene has a transit peptide
sequence attached so it is targeted to the plastid, where geranylgeranyl
diphosphate formation occurs. The bacterial crt1 gene was an important
inclusion to complete the pathway, since it can catalyze multiple steps in the
synthesis of carotenoids, while these steps require more than one enzyme in
plants.
The end product of the engineered pathway is lycopene, but if the plant
accumulated lycopene, the rice would be red. Recent analysis has shown the
plant's endogenous enzymes process the lycopene to beta-carotene in the
endosperm, giving the rice the distinctive yellow colour for which it is named.
The original golden rice was called SGR1, and under greenhouse conditions it
produced 1.6 g/g of carotenoids.

Golden rice has been bred with local rice cultivars in

the Philippines, Taiwan and with the American rice cultivar 'Cocodrie'. The first
field trials of these golden rice cultivars were conducted by Louisiana State
University Agricultural Center in 2004.
Field testing will allow a more accurate measurement of the nutritional value of
golden rice, and will enable feeding tests to be performed. Preliminary results
from the field tests have shown field-grown golden rice produces 4 to 5 times
more beta-carotene than golden rice grown under greenhouse conditions.
In 2005, a team of researchers at biotechnology company, Syngenta, produced a
variety of golden rice called "Golden Rice 2". They combined the phytoene
synthase gene from maize with crt1 from the original golden rice. Golden rice 2
produces 23 times more carotenoids than golden rice (up to 37 g/g), and
preferentially accumulates beta-carotene (up to 31 g/g of the 37 g/g of
carotenoids). To receive the Recommended Dietary Allowance (RDA), it is
estimated that 144 g of the most high-yielding strain would have to be
eaten. Bioavailability of the carotene from either variety has not been tested in
any model.
In June 2005, researcher Peter Beyer received funding from the Bill and
Melinda Gates Foundation to further improve golden rice by increasing the
levels of or the bioavailability of pro-vitamin A, vitamin E, iron, and zinc, and
to improve protein quality through genetic modification.[10]

2.Several food crops are now being developed with enhanced vitamin E

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and

some cyanobacteria

Deficiency of vitamin E leads to muscle weakness, anemia sight problem

blurred vision.

In the tocopherol biosynthesis pathways, -tocopherol methyltransferase ( -

TMT) is the final enzyme that catalyzes the conversion of -tocopherol into -
tocopherol, the most bioactive species of tocopherol gamma-tocopherol methyl
transferase (gamma-TMT) gene is over expressed in several food crops like
soybean

3. Starch production in Potato

Starch is the predominant ingradient of the main food and feeds commodities
such as cereal grains and potato tubers.

It is also predominant in root tubers such as sweet potato yam and cassava

Starch is very important in food industry where it is mainly derived from maize
grains and potato tubers.

Role in fast food industry and the quality of the starch may affect the quality
and their fried product.

Starch synthesis takes place in chloroplast in leaves and plastids in storage

organs

Linear chain starch amylase synthesis

1.Glucose 1-Phosphate to ADP Glucose with the help of the enzyme ADP
glucose pyrophosphorylase (ADPGPP).

2. These glucose residues were connected in to 1-4 linked linear chain by Starch
syntase enzyme (SS).
Difference in amylase is due to the changes or isozymes of ADPGPP and SS

3. A third enzyme of starch branching enzyme SBE that hydrolyse the 1-4
linkage within the chain and catalyse the formation of 1-6 linkage between the
cut glucan chain and other glucose residues.

Such cross linkage produced branched amylopectin starch.

Normally Amylose make up of 11-37 % of the total storage starch

But great variations occur amylase and amylopectin ratio.

Transgenic approach was attepted to increase or decrease the starch

content or Amylose and amylopectin ratio
Mosanto did extensive research they used the ADPGPPase mutant Gene glgC16
isolated from bacteria were transferred to potato with tuber specific promoter
(patatin).

Produced tuber with 60% more starch. The total fresh weight of potato tuber
was similar with control. But the starch content and dry weight of potato
increased.

Thus the preservation in cold storage and frying quality was improved. The
sprouting of this tubers delayed.

Dutch investigators used antisense RNA technology to reduce the amylose

content by reducing the activity of SS enzyme. This enzyme is essential for
amylose synthesis

Introduction of glg-A gene in to potato reduced starch and amylose amylopectin

ratio 30-50% in potato.

Potato is the most important non-cereal food crop for human consumption and,
therefore, the need to improve its nutritional quality cannot be overemphasized.

Expression of the AmAl gene isolated from amaranth (Amaranthus

hypochondriacus L.) in potato led to a significant increase in most essential
amino acids as well as in higher protein content in tubers compared with non
transgenic plants

Production of potent anti-oxidants including lycopene has been increased

through transgenic over expression of relevant enzymes in tomatoes
3.Genetic modifications of plants to generate useful Products

Biochemistry of Fruit Ripening and Softening

Fruit ripening and softening is a highly controlled developmental process.

Firmness of fruit is a function of the properties of the cell wall (Cellulose fibers,
Pectins, Hemicellulose and Proteins).

Various enzymes that degrade specific components of the cell wall are
synthesized during fruit ripening (i.e. Cellulase -breaks down cellulose;
Polygalacturonase
(PG) and pectin methyltransferase (PME) break down cross-linking pectin
molecules).

To delay fruit ripening, slow down the ethylene response of the ripening
pathway. This approach or technique could be used on any climacteric fruit.

Strategies to improve postharvest shelf life

There are three main ways to improve post-harvest shelf life:

1)By blocking the expression of genes (such as PG and PME) that are induced
in response to ethylene
(i.e. Flavr Savr tomato ).
2) By blocking ethylene synthesis.
3) By blocking the reception to ethylene.
Expression of a dominant mutant ethylene receptor

Using Antisense technique to Reduce PG Expression in Tomatoes

1. Clone the PG gene from tomato and construct a chimeric gene to express
antisense RNA for PG in the fruit.
A promoter that is highly active in fruits - The ORF of PG, flipped so that
antisense RNA will be transcribed A transcription terminator
2. Transform to produce transgenic fruit with this antisense RNA gene.
3. Evaluate these transgenic plants.
Altering Fruit Ripening with Antisense RNA

Polygalacturonase (PG) is an enzyme that breaks down pectin in ripening fruit

walls.
Plants with an antisense PG transgene produce less PG.
-Walls soften more slowly.

Using Antisense technique to Reduce PME Expression in Tomatoes

PME is involved in metabolism of pectins in the cell wall. Pectin in mature

green fruits are long polymers.
During fruit ripening PME breaks down the long polymers to short polymers.

The same antisense RNA technique was used to reduce the PME expression in
fruits.
Longer polymers increased the viscosity of juice from transgenic low-PME
fruits than the control samples.

Antisense PG and PME Transgenic Fruits

Antisense PG technique used by Calgene to produce FLAVR SAVR tomatoes

failed to produce desired post-harvest quality.

Antisense PME technique was good for viscosity of juice and was used to make
good quality tomato paste faster but did not improve shelf life.

In both cases, the reason may be that nothing was done to reduce ethylene
production.
Using Antisense technique to Modify Ethylene Responses in Transgenic Plants

Ethylene, a natural hormone produced by some fruits as they ripen, promotes

additional ripening of produce exposed to it.

Damaged or diseased apples produce high levels of ethylene and stimulate the
other apples to ripen too quickly.

As the fruits ripen, they become more susceptible to diseases.

Some climatic fruits as: avocados, papayas, passion fruit, peaches, persimmons,
plantains, prunes, tomatoes, etc. are highly sensitive to ethylene

Ethylene "producers" should not be stored with fruits, vegetables, or flowers

that are sensitive to it.
The result could be loss of quality, reduced shelf life, and specific symptoms of
injury.
Most climacteric fruit picked green, shipped to market and treated with ethylene
before sale at wholesale level.

The autocatalytic synthesis of the gaseous hormone ethylene regulates the

expression of specific genes involved in tomato ripening.
In climacteric fruits like apple the exponential increase in ethylene production
coincides with a rise in respiration and correlates with the development of FFC
(Fruit Flavor Composition).

Biosynthesis of Ethylene

Biosynthesis of ethylene occurs in two enzymatic steps:

S-adenosyl methionine is converted to ACC (1- aminocyclopropane-1-
carboxylic acid) by ACC synthase (ACS).

ACC is converted by ACC oxidase to ethylene.

SAM
ACC synthase

ACC

ACC oxidase

Ethylene

Biotechnology to Modify Ethylene Responses in Transgenic Plants

Ethylene changes in gene expression initiation of ripening or senescence
Ethylene binds to receptor signal transduction pathway changes in gene
expression
Transgenic plants with ethylene response blocked:
- Fruit will not ripen, even if exposed to ethylene (i.e. Apple).

Ethylene Suppressed Transgenic Plants Approach

Transgenic apple plants were produced in which the genes coding for key
enzymes of ethylene biosynthesis were silenced. (i.e. ACC synthase )

Ethylene suppressed fruits were firmer and displayed increased shelf-life.

Sugar and acid accumulation was not different from controls.

Terminator Technology
'Terminator,' officially named the Technology Protection System (TPS),
incorporates a trait that kills developing plant embryos, so seeds cannot be
saved and replanted in subsequent years.

Scientists from the Agricultural Research Service, a subunit of the US

Department of Agriculture (USDA-ARS), and Delta and Pine Land Company, a
company that develops cotton cultivars, jointly developed the system and on
March 3, 1998, were awarded a jointpatent (number 5,723,765) entitled Control
of Plant Gene Expression.

Mechanism involved in terminator technology

The patented method for terminator technology is based on a gene that produces
a protein that is toxic to the plant and therefore, does not allow the seed to
germinate.

One such gene indicated in the patent, is ribosomal inactivating protein (RIP)
gene, which if expressed, does not allow protein synthesis to take place.

The gene is placed under the control of LEA promoter permitting RIP to
express only during late embryogenesis, thus affecting only the embryo
development.

This gene (RIP gene) will not express in the first generation, because its
expression is blocked through the use of a spacer or a blocking sequence
between the promoter and the lethal RIP gene.

On either side of the spacer are placed specific excision sequences that are
recognized by a recombinase enzyme (CRE/LOX system from a bacteriophage),
whose function is to excise the spacer or the blocking sequence.

The second gene encoding recombinase is placed behind another

promoter/operator, specific for a repressor encoded by a the third gene, which is
a repressor gene. The genetic elements, as above, differ for pure lines and
hybrid seed production systems (see below).
Technology for pure lines seed production
In self-pollinated crops, where pure lines are used as cultivars, the promoter for
the above recombinase gene is repressor specific and is used by a specific
repressor protein encoded by the third gene, which is a repressor gene.

The repressor gene can be switched off using a stimulus in the form of a
chemical, a heat shock, etc. Due to the stimulus, the repressor gene is switched
off and CRE/LOX (recombinase gene) is switched on. This will lead to the
production of recombinase enzyme, which will excise the blocking sequence or
the spacer which blocked the expression of the terminator gene (e.g. RIP).

Due to excision of the blocking sequence, the promoter will come to lie adjacent
to the terminator gene, which will now be expressed and will kill the developing
embryos.

In actual commercial production and sale of seeds of pure lines as above, the
crop for commercial seed production will be grown by the breeder or seed
company without chemical treatment.

The harvested seeds from this crop, will be treated with the chemical, before it
is sold to the farmer, so that in crops grown by the farmer, the toxic substance is
produced at the time of embryo development, and the embryos die or fail to
develop (Figure 1).

A seed thus produced will carry the endosperm, but not the embryo, so that it
can be used or sold as grain, but cannot be used for sowing. Tetracycline is one
such chemical trigger that can be used for treatment of the seeds before selling
them in the market.

Technology for hybrid seed production

In case of hybrid seed production, a different strategy, utilizing only two genes
(terminator and recombinase), one in each of the two parents of the hybrid are
used and no repressor gene is needed.

One of the parental lines contains the recombinase gene, which becomes active
only after germination, and the other parent contains the lethal (terminator) gene
separated from its promoter by a spacer (blocking sequence).

The hybrid progeny, which is the technology protected hybrid seed bought and
planted by the farmer, thus contains both the elements of the system in every
cell. The recombinase, expressed right after germination, excises the spacer
blocking sequence bringing the promoter and lethal gene together.
Since the promoter is embryo specific, the lethal gene does not express till seed
development starts. During seed development, the lethal gene expresses during
late embryogenesis and kills the embyo (Figure 2). Thus the seeds harvested
from the first generation hybrid crop will be normal in all essential respects,
except that they will not germinate if sown as a crop.

Potential Benefits of the Technology Protection System

Companies may develop improved crop varieties

The USDA-ARS and Delta and Pine Land Company say the technology will
encourage seed companies to pursue variety development more vigorously,
especially in "minor crops" for which seed companies typically do not expend a
large amount of money in research. If a seed company knows it will be able to
"protect its investment" in the development of new varieties, including
transgenics, by preventing farmers from saving seed and replanting from year to
year, more effort will be put into the development of improved varieties.

Potential Risks of the Technology Protection System

Pollen from TPS plants may kill neighboring crop seeds

Pollen from TPS plants may fertilize non-TPS crops in neighboring fields,
resulting in the unintentional death of seeds in the non-TPS crop. The USDA-
ARS and Delta and Pine Land Company say the risk of this will be low because
TPS technology is intended for highly self-pollinating crops; therefore,
pollination from adjacent fields is unlikely. However, research is underway to
develop TPS crops that do not carry any transgenic genes on the pollen,
preventing unwanted transfer of the trait.

Pollen from TPS plants may kill neighboring wild plant seeds
While this can be viewed as a benefit in preventing the spread of transgenic
traits to wild populations, it may have a negative impact on the ability of wild
populations to maintain themselves. Depending on the propensity of the crop for
crossing to wild populations, some rare or endangered plant populations might
be threatened by the presence of TPS crops. Case-by-case consideration will be
needed.

Small farmers may suffer economic harm

Small farmers who have depended on the ability to replant seeds from year to
year may be hurt because they will have to buy new seeds each year.
The method used to kill the seeds may harm other organisms
The TPS patent notes that the ribosome inhibitor is the preferred agent for
killing seeds because it is not lethal to people who might eat the seeds
The chemical treatment may have negative impacts on the environment
The patent authors envision using the antibiotic tetracycline to trigger the TPS
system. Large-scale use of this antibiotic, used to treat human diseases, may
cause an increase in antibiotic resistance in bacteria in the environment

Superweeds
Super weeds refers to unwanted plant species which have become resistant to
herbicides. Herbicides are chemicals used to kill unwanted plants. This is a
potential problem with any type of herbicide, but has especially become a
discussed issue within the context of genetically modified (GM) agricultural
crops.

The leading US (and global) company developing GM crops is Monsanto.

Many of their best-selling crops are designed to be resistant to a herbicide called
glyphosate ("Round-Up"). Glyphosate is sprayed on the field before planting,
kills everything (almost), degrades rapidly and soon the glyphosate-resistant
crop can be planted and it will grow well.

The resisant crop sometimes breeds with wild but closely-related species,
creating weeds that were never much of a problem before (superweeds). This is
especially likely to happen when a glyphosate-resistant crop is grown on the
same patch of land year after year. Moreover, the GM crop may itself become a
superweed, bred as it has been, to be resistant to herbicides if the farmer wants
to plant something different in later years.

Example actual or potential superweeds are rye-grass in Australia, soybeans in

North American and many wild cousins of oilseed rape in Britain. Anonymous

Health care products in Transgenic plants

The advent of genetic engineering in the early 1970s and its application to
plant biology revolutionized agriculture and contributed to the dramatic growth
of the biotechnology industry.

The exploitation of plant biotechnology to produce diagnostic and therapeutic

products has become a well-recognized and important field of
biopharmaceutical science with promising economic potential.
whole plants and plant cell cultures are now considered as viable and
competitive systems for large-scale production of industrial, pharmaceutical
recombinant proteins and secondary metabolites.

The first transgenic plants were reported in 1983. Since then, many recombinant
proteins have been expressed in several important agronomic species of plants
including tobacco, corn, tomato, potato, banana, alfalfa and canola

Recombinant protein production using transgenic plants as bioreactors is likely

to be more economical than alternative systems, especially for large-scale
needs. Factors in favor of plant systems as sources of animal derived proteins,
compared with other conventional methods, include:

The potential for large-scale, low-cost biomass production using

agriculture.
Low risk of product contamination by mammalian viruses, blood-borne
pathogens, oncogenes and bacterial toxins.
The capacity of plant cells to correctly fold and assemble, not only
antibody fragments and single chain peptides, but also full-length
multimeric proteins.
Low downstream processing requirements for proteins administered
orally.
Elimination of the purification requirement when the plant containing the
recombinant proteins is edible, such as potatoes.
The ability to introduce new or multiple transgenes by sexual crossing of
plants.
The avoidance of ethical problems associated with transgenic animals.
Formulated in seeds, plant-made enzymes have been found to be an
extremely convenient method for reducing storage and shipping costs, for
an indefinite amount of time, under ambient conditions.
Production size is flexible and easily adjustable to the needs of changing
markets.

Plants are also capable of synthesizing and assembling virtually any kind of
antibody molecule, ranging from the smallest antigen-binding domains and
fragments, to full length, and even multimeric antibodies.

There are, however, potential issues of concern for plant protein production:

Allergic reactions to plant protein glycans and other plant antigens.

 Plant and product contamination by mycotoxins, pesticides, herbicides
and endogenous metabolites.
Regulatory uncertainty, particularly for proteins requiring approval for
human drug use (Doran 1999).

antibodies and antibody fragments, pathogen-derived protein antigens and

subunit vaccine epitopes, hormone- and neuropeptides,enzymes of therapeutic
and nutritional significance and secondary metabolites with pharmacological
properties of medicinal value

Production of whole antibodies and antibody fragments in plants

Antibodies are protein molecules formed by the B lymphocytes of the vertebrate

immune system that recognize and bind to specific molecular epitopes (termed
antigens)

The role of the antibody is to neutralize the antigen and target it for removal
from the cell.

The first reports describing the successful expression of antibodies in transgenic

plants occurred in the late 1980s and early Since that time, the combination of
two rapidly advancing technologies (immunology and plant genetic
engineering), has resulted in the expression of a diverse range of antibodies
(often referred to as plantibodies) in numerous vascular plant and green algal
species

Several research groups have successfully expressed active, full-length

antibodies in plants by targeting the antibodies to the apoplastic space (i.e., the
space between adjacent plant cells) via the endoplasmic reticulum

two different approaches has been employed to produce biologically active

whole antibodies in plants:

transformation of the heavy and light-chain genes separately into plants,

followed by cross-pollination of the two transgenic parents to yield F1
individuals carrying both the heavy and light chain gene and cotransformation
of the heavy and light chain genes on a single expression cassette

cross-pollination strategy, it is also possible to produce a wide range of novel

antibodies by combining plant lines carrying different sets of modifications in
either the heavy or light chains IgG 6D4 antibody in plants have been produced
using this style
Although tobacco leaf has been the most popular expression system for
recombinant antibodies, several groups have explored the use of other plants
and plant seeds for use in expression and large-scale production.

For example, Stoger successfully expressed the scFv antibody (ScFvT84.66)

against carcinoembryonic antigen (CEA), in the leaves and grain of transgenic
rice and wheat. High levels of antibody production could also be achieved in
transgenic potato tubers

Corn seeds have shown particular utility in the production of high levels of
secretory immunoglobulin A (sIgA), the most abundant antibody class produced
by the body (>60% of total immunoglobulin) and secreted onto mucosal
surfaces to provide local protection from toxins and pathogens.

CaroRxTM is an anti-Streptococcus mutans secretory immunoglobulin A (sIgA)

used for the prevention of dental cariesIn the human oral cavity, the secretory
antibody survived for up to three days, compared with one day for the IgG
antibody. The plant secretory antibody (Tobaco plant) afforded specific
protection in humans against oral streptococcal colonization for at least 4
months

Planet Biotechnology Inc. completed United States Food and Drug

Administration (FDA)-approved phase I/II confirmatory clinical trials at the
School of Dentistry at the University of California,

Avicidin is a full-length antibody specific for EpCAM (a marker of colorectal

cancer) developed jointly by NeoRx and Monsanto.

a mouse-derived antibody that is in clinical trials to treat patients with solid

tumors such as lung, prostate, colon and ovarian cancers that no longer respond
to standard therapies

NeoRx represented the first time that such a protein produced in plants has been
tested in humans. Unfortunately, Phase II trials were discontinued after
exhibited significant gastrointestinal side effects

T84.66 is a monoclonal antibody that has been used successfully for in vivo
imaging and diagnosis of human colorectal carcinoma

The full length IgG has been transiently expressed in tobacco by agroinfiltration
and the scFv has been expressed in transgenic tobacco, pea rice and wheat
The Anti-HSV IgG recognizes herpes simplex virus 2 (HSV-2). Zeitlin (1998)
expressed humanized anti-herpes simplex virus 2 (HSV-2) monoclonal antibody
in soybean

Anti-human chorionic-gonadotropin (HCG) HCG is synthesized and secreted

soon after fertilization and traditionally it has been used as an index for
pregnancy a chimeric full-size IgG, an scFv fragment, and a diabody derived
from the original anti-hCG murine mAb PIPP and expressed them in tobacco
plants.

Studies of efficacy have shown that the full-size anti-hCG antibody was 1000
times more active than either the scFv fragment or diabody derivative. These
could potentially be used for pregnancy detection (emergency)

Monsanto Protein Technologies, estimated that an animal cell culture

manufacturing facility designed to produce 500 kg of MAb per year of the type
(a single product facility) currently used by the pharmaceutical industry would
require up to $450 million dollars in capital and would take 47 years to build
and approve.

In contrast, the same product could be grown on 500 acres of corn, purified in a
facility costing $80 million and requiring three to five years to build and
approve. Cost per gram of MAbs by traditional means is $350$1200 per gram
(depending on scale). Corn would cost $80$250 per gram (depending on
scale).

Currently, more than 200 novel antibody-based potential products are in clinical
trials worldwide, Vaccine Production

Vaccination can be accomplished by introducing the antigen parenterally into

the body or bloodstream,

By comparison, parenteral immunization is more efficient since oral

immunization appears to require a considerably large amount of subunit or
soluble antigen (mg versus lg amounts) to elicit a response (De Aizpurua and
Russell-Jones, 1988). For this reason at the present time there is considerable
interest in exploiting the ability to overexpress antigenic proteins to human
disease-causing agents in transgenic plants for use in vaccine production.

Edible vaccines

Subunit vaccines consist of specific macromolecules that induce a protective

immune response against a pathogen.
These vaccines are the consequence of advances in molecular biology enabling
the location of antigens capable of invoking a protective immune response
(vaccinogen), the isolation of the corresponding gene(s), and the production of
the vaccinogen in an expression system.

Subunit technology has improved existing vaccines and has circumvented some
limitations of traditional vaccine production.

The specific composition of subunit vaccines increases vaccine safety by

circumventing the need to use live viruses or microbes and has thus made them
the preferred approach for vaccine manufacturers

Unfortunately, traditional subunit vaccines are expensive to produce and not

heat stable (making them reliant on the expensive, often capricious and
destination limited series of refrigeration steps, sometimes referred to as the
cold chain, en route from manufacture to vaccination).

This limits their availability and use in the low-funded health care systems of
developing countries. Production of vaccines in plants eliminates some current
impediments. The simplistic requirement of plants for sunlight, water and
minerals makes them an inexpensive means
of correctly processing and expressing proteins that can be quite complex.

Expression of vaccines in plant tissues eliminates the risk of contamination with

animal pathogens, provides a heat-stable environment, and enables oral
delivery, thus eliminating injection-related hazards.

The idea for transgenic plant-derived vaccines originated in the early 1990s. At
the time, Charles Arntzen and his colleagues envisaged a cost-effective vaccine
production system with a safe and efficacious delivery system through the use
of plants specifically engineered to deliver safe subunit preparations of
candidate antigens for major diseases afflicting developing and developed
nations

The first demonstration of vaccine antigen expression was reported in 1990

when R.I. Curtiss and C.A. Cardineau expressed the Streptococcus mutans
surface protein antigen A (SpaA) in tobacco (Curtiss and Cardineau, 1990).
After feeding the transgenic tobacco tissue to mice, the researchers observed
that a mucosal immune response was induced in response to the SpaA protein.

Subsequently C. Arntzen and his collaborators provided proof of concept for

what is now referred to as edible vaccines by showing that the surface antigen
of Hepatitis B could be synthesized in transgenic plants and that animals fed the
transgenic plant materials containing the immunogenic protein developed a
specific immune response against the recombinant antigen

Since these initial reports, numerous plant species have been used for antigen
expression, including tobacco, potato, tomato, banana, corn, lupine, and lettuce

Hepatitis B -Tobacco Lettuce, Rabies virus -Tomato, Vibrio cholerae -Potato,

Foot and mouth disease- Arabidopsis , Respiratory syncytial virus -Tomato,
Measles virus (MV)- Tobacco, Heat-labile enterotoxin Corn, Cholera,
enterotoxigenic -E. coli- Potato Cholera, enterotoxigenic E. coli -Potato
Peptides, proteins and enzymes of pharmacological value
Important peptide, proteins and enzymes have been produced in transgenic
plants, offering numerous biological and economical advantages as compared to
other industrial systems

For example, the cost of producing large numbers of recombinant proteins in

plants and generating large amounts of biomass as a starting point for protein or
enzyme purification is relatively inexpensive compared to the cost of large scale
microbial fermentation and animal cell culture.

Plants have been tested for a range of therapeutic proteins to be used either
directly in foods or after purification. Expression of milk proteins,such as
lactoferrin and beta-casein, in plants may contribute the therapeutic values of
these protein to other food products. As a food source, casein supplies amino
acids; carbohydrates; and two inorganic elements, calcium and phosphorus.

Recently, Staub (2000) reported the production of correctly processed human

somatotrophin (hST) (growth hormone) in transgenic tobacco plants, with an
expression level in chloroplast of up to 7% total soluble protein. The growth
hormone hST is used for the treatment of hypopituitary dwarfism in children,
and has possible future uses in treating Turner syndrome, chronic renal failure
and HIV wasting syndrome

a-Trichosanthin- Tobacco, Hirudin -Brassica seeds, a-Interferon- Rice,

Erythropoietin- Tobacco, Avidin -Maize, Lactoferrin and beta-casein- Potato,
Phytase- Canola, Human a-antitrypsin- Rice

Secondary metabolites of pharmacological value

The plasticity of plant metabolic activity is most evident in the wide variety of
secondary metabolites accumulated by plants in their leaves, roots, and other
organs

Within recent times, many plantderived products for treating human disorders
have reached the market place as useful drugs including atropine, hyoscyamine,
scopolamine, taxol (anticancer), vinblastine/vincristine, artemisinin
(antimalarial), reserpine (antihypertension), and quinine (antimalaria).

The ability to genetically engineer plant genomes has allowed for the direct
manipulation of plant metabolism and the potential for manipulating the content
and nature of plant secondary metabolites of commercial value. Plants are now
being considered as potential factories for the production of a variety of useful
compounds
Secondary Metabolite production

For centuries, mankind is totally dependent on plants as source of

carbohydrates, proteins and fats for food and shelter.

In addition, plants are a valuable source of a wide range of secondary

metabolites, which are used as pharmaceuticals, agrochemicals, flavours,
fragrances, colours, biopesticides and food additives.

Over 80% of the approximately 30,000 known natural products are of plant
origin

The number of known chemical structures is estimated to be nearly four fold

greater than that in the microbial kingdom.

Worldwide, 121 clinically useful prescription drugs are derived from plants

Rational engineering of secondary metabolic pathways in plants requires a

thorough knowledge of the whole biosynthetic pathway and a detailed
understanding of the regulatory mechanisms controlling the onset and the flux
of the pathways.

Such information is not yet available for the vast majority of secondary
metabolites, explaining why only limited success has been obtained by
metabolic
engineering.

There are several strategies that can be used to enhance the production of
desired pharmaceuticals by genetic engineering

(1) Decrease the catabolism of the desired compound.

(2) Enhance the expression or activity of a rate-limiting enzyme.
(3) Prevent feedback inhibition of a key enzyme.
(4) Decrease the flux through competitive pathways.
(5) Enhance expression or activity of all genes involved in the pathway.
(6) Compartmentalization of the desired compound.
(7) Conversion of an existing product into a new product.

Only a fewof these strategies have been successfully demonstrated so

far.
The gain-of-function and loss-of-function strategies have been adopted with
individual genes.

The discovery of transcription factors that can regulate the entire pathway has
opened new avenues towards controlled production of secondary ymetabolites,
for example, in cultivated plant cells.

The over expression of transporters in cultivated plant cells has also shown the
potential to transport toxic compounds from inside the plant cell to extracellular
space.

Recently, the simultaneous over expression of two genes encoding the rate-
limiting upstream enzyme putrescine N-methyltransferase (PMT) and the
downstream enzyme hyoscyamine 6b-hydroxylase (H6H) of tropane alkaloid
biosynthesis resulted in the highest scopolamine production ever obtained in
cultivated hairy roots

The potential of metabolically engineering plant derived secondary metabolites

is high, and has been well documented by modifying anthocyanin and flavonoid
pathways leading to changes in flower color or increased levels of antioxidative
flavonol production in tomato.

However, to date, there have been few successes in modifying pathways to form
pharmaceutically important compounds.

The introduction of a gene encoding hyoscyamine-6b-hydroxylase, previously

identified in Hyoscyamus niger, into Atropa belladonna resulted in the
accumulation of the high-value tropane alkaloid scopolamine, previously found
only in trace amounts in A. belladonna.

Virtually all hyoscyamine was converted to scopolamine. This was the first
successful example of engineering an important medicinal plant to produce a
valuable end product. An even more drastic effect was obtained when the same
gene was over expressed in Hyoscyamus muticus hairy roots

In this case, not only were large amounts of scopolamine produced but also high
levels of hyoscyamine accumulated in the hairy roots.

Another example is the observation that antisense-mediated down regulation of

putrescine N-methyltransferase in transgenic tobacco plants resulted in a
concomitant reduction in nicotine content but surprisingly also in elevated
levels of the secondary metabolite anatabine
Part of the terpenoid indole alkaloid biosynthesis in Catharanthus roseus is
under the control of ORCA3, a jasmonate responsive APETALA2 (AP2)-
domain transcription factor. Constitutive overproduction of ORCA3 in cell
cultures resulted in enhanced expression of several biosynthetic genes as well as
enhanced accumulation of terpenoid indole alkaloids

Overexpression of the maize transcription factors LC and C1 in tomato fruits

upregulated the flavonoid pathway and led to the accumulation of the
flavonol kaempferol and, to a lesser extent, the flavanone naringenin.

Anthocyanin biosynthesis in tomato is controlled by a MYB transcription

factor, denominated ANT1. Over expression of ANT1 caused anthocyanin
accumulation and the upregulation of genes encoding proteins in the early and
late steps of anthocyanin biosynthesis as well as genes implicated in
anthocyanin glycosylation and transport into the vacuoles

Secondary metabolites in cell and tissue cultures are usually stored intra
cellularly, for example, in vacuoles, and transporters probably play an important
role in the
sequestration of secondary metabolites.

An important question is: when the concentration of a highly toxic secondary

metabolite is increased by genetic engineering, does its intrinsic toxicity
become a limiting factor?

Recent experiments suggest that this might be the case. Nicotine and also other
alkaloids are highly toxic to plant cells but over expression of the yeast ABC
transporter PDR5 in transgenic tobacco cells was recently demonstrated
to decrease the cellular toxicity
 Applications of transgenic
Animals
Creating Transgenic Animals

Once a suitable transgene is available the

standard scenario for the creation of a
transgenic animal by nuclear microinjection is
as follows:
1. The transgene is injected into fertilized egg
cells. Just after fertilization, the egg contains its
original female nucleus plus the male nucleus
from the successful sperm.

These two pronuclei will soon fuse together.

Before this happens, the DNA is injected into
the male pronucleus, which is larger and
therefore a better target for microinjection..

2. The egg is kept in culture during the first

few divisions of embryonic development.

3. The engineered embryos are then implanted

into the womb of a female animal, the foster
mother. Here they develop into embryos and, if
all goes well, into newborn animals.

4. Some of the baby animals will have the

transgene stably integrated into their
chromosomes. In others the process fails and
the transgene is lost. Those that received
the transgene and maintain it stably are called
founder animals.

A male and female are mated together to form a new line of animals carrying
two copies of the transgene.

Note that the founder animals contain only a single copy of the transgene
on one chromosome, and are heterozygous for the transgene. When two such
founder animals are bred together, 25% of the progeny will get two copies
of the transgene and will be homozygous, 25% will get zero copies, and the
remaining 50% will get one copy. Homozygous transgenic animals are most
useful because if these are further interbred, all of their descendants will get two
copies of the transgene.

Embryonic stem cells may also be used to generate transgenic animals.

Stem cells are the precursor cells to particular tissues of the body.
Embryonic stem cells are derived from the blastocyst, a very early stage of
the embryo, and retain the ability to develop into any body tissue, including
the germline.

Embryonic stem cells can be cultured and DNA can be introduced as for any
cultured cell line.

Engineered embryonic stem cells are then inserted into the central cavity of
an early embryo at the blastocyst stage. This creates a mixed embryo and results
in an animal that is a genetic chimera consisting of some transgenic
tissues and others that are normal.

If the host embryo and the embryonic stem cells are from different genetic
lines with different fur colors, the result is an animal with a patchwork coat.
This allows the transgenic sectors of the animal to be identified easily. This
chimeric founder animal must then be mated with a wild-type animal. If the
embryonic stem cells have contributed to the germline, then the coat color
characteristic of this cell line will be transmitted to the offspring.

In mice black (recessive) and agouti (dominant) coat colors are often used. The
embryonic stem cells are usually taken from an agouti line, because the
dominant fur color enables the transgenic cell lines to be tracked easily.

Both the embryonic stem cells and the host embryo are usually taken from
males because the resulting male chimeras can father many children when
crossed with wild-type females.

knockout mouse
A knockout mouse is a genetically engineered mouse in which researchers
have inactivated, or "knocked out," an existing gene by replacing it or
disrupting it with an artificial piece of DNA. The loss of gene activity often
causes changes in a mouse's phenotype, which includes appearance, behavior
and other observable physical and biochemical characteristics.

Knockout mice are important animal models for studying the role of genes
which have been sequenced but whose functions have not been determined. By
causing a specific gene to be inactive in the mouse, and observing any
differences from normal behaviour or physiology, researchers can infer its
probable function.

Applications of Knock out mice

Experimental approaches to modulate gene expression in vivo

targeted gene disruption by homologous recombination in embryonic stem cells have been
employed widely to produce animal models with specific gene deletions. Many of these
models are quite useful for nutritional and metabolic research. Therefore, this review will
focus on the use of this technique.
Utility of gene knockout mice to explore physiologic functions and produce animal
models for human disease

gene knockout mice can be produced as an animal model for human diseases. These gene
knockout mice can then be used to explore potential intervention strategy, including
pharmacological and genetic therapy approaches, for treatment of specific metabolic diseases.

A second utility for the production of knockout mice is to explore physiological function and
significance of specific genes.

Potential species differences in extrapolating data from knockout mouse studies to

human health issues

Although the generation of gene knockout mice is clearly a valuable tool for producing a
rodent model of human disease and to evaluate physiologic functions of specific genes, it
must be noted that not all the knockout mice will necessarily produce a predictable phenotype
similar to those observed in human subjects.

species differences in metabolism and physiology may also explain some of the discrepancies
in these results.

Importance of genetic background in evaluating gene defects in physiology and

pathophysiology

results showed a direct correlation between AGT gene copy number and arterial blood
pressure. The latter study revealed that the plasma AGT level in their agt(+/) animals was
35% of normal, significantly less than the 50% level expected from their genotype. Thus,
their results clearly indicated the involvement of a second gene. An interpretation that is
consistent with the results with agt(/) mice with mixed genetic background.

Candidate gene approach to identify proteins involved with a complex physiological

trait

Gene knockout technology also offers the opportunity for testing the physiological
importance of specific genes and gene products in a complex physiological trait. For
example, dietary cholesterol absorption efficiency has been shown to be regulated by
multiple genetic factors (Carter et al. 1997). The genes and gene products involved with the
cholesterol absorption process can only be inferred from cell culture studies as well as studies
with metabolic inhibitors. However, results of studies using the latter approaches may not
always reflect physiological situations and candidate genes identified from these studies need
to be verified in vivo. The cholesterol esterase studies described previously clearly illustrated
the utility of gene knockout mice to test the physiological importance of candidate genes.

Animal models of human disease

Transgenic animal disease models are animals that have been genetically altered
to have traits that mimic the symptoms of specific human pathologies.
The disease models are needed so that we can better understand the disease for treatment.
Many animals do not normally exhibit the equivalent of certain human diseases. So a human
transgene specific to the disease needs to be expressed in the animal.

This allows for pathological characteristics in the animal so that it can be studied. Animal
disease models are very useful in that they allow us to screen drugs that may be harmful or
have bad side affects.

Once the therapeutic agents have been discovered and tested, human cells may then be tested,
followed by human test subjects in clinical trials.

But because it is not ethical or safe to perform the initial tests in humans, we use transgenic
animals.

Animal pharming

Since its inception 20 years ago, the animal pharming industry has promoted transgenic
animals as a cost-effective method of biopharmaceutical production. However, it took until
2006 for the first therapeutic product to gain regulatory approval.

The main reason to express a particular protein in either animals or cultured animal cells,
rather than bacteria, plants or yeast, is because post-translational processing is required for
bioactivity.

This applies to many of the most important biomedical products, such as blood clotting
factors and antibodies and there are currently no alternative means of expression.

Almost any protein currently produced in mammalian cell culture could also be produced
successfully in milk.

Alternatives to milk where products are strictly sequestered, e.g. urine or avian eggs, may
overcome this limitation in future.

The first transgenic animal, a mouse, was produced in 1981. In an effort to determine which
genes were involved with cancer, a gene was inserted into the mouse that made it susceptible
to cancer. In 1985, the first transgenic farm mammal was produced, a sheep called "Tracy".
Tracy had a human gene that expressed high levels of the human protein alpha-1-antitrypsin.
The protein, when missing in humans, can lead to a rare form of emphysema.

Farm animals for pharmaceutical production

The production of human therapeutic proteins by recombinant bacteria or cell cultures
have several limitations.

They are only suitable for simple proteins, the amount of protein produced is limited, and
post-translational modifications are often incorrect leading to immune reactions against the
protein.
In addition, the technical prerequisites are challenging and production costs are high.

Farm animals such as cattle, sheep, goats, pigs and even rabbits have several significant
advantages for the production of recombinant proteins over other systems, including their
potential for large-scale production, correct glycosylation patterns and post-translational
modifications, low running costs, rapid propagation of the transgenic founders and high
expression stability.
These attractive perspectives led to the development of the Animal pharming concept

The most promising site for production of recombinant proteins is the mammary gland, but
other body fluids including blood, urine and seminal fluid have also been explored

The mammary gland is the preferred production site mainly because of the quantities of
protein that can be produced and the ease of extraction or purification of the respective
protein.

Based on the assumption of average expression levels, daily milk volumes and purification
efficiency, 5400 cows would be needed to produce the 100 000 kg of human
serum albumin (HSA) that are required per year worldwide, 4500 sheep would be required
for the production of 5000 kg a-antitrypsin (a-AT), 100 goats for 100 kg of monoclonal
antibodies, 75 goats for the 75 kg of antithrombin III (ATIII) and two pigs to produce 2 kg
human clotting factor IX.

proteins have been produced by targeting expression to the mammary gland via mammary
gland-specific promoter elements. Proteins were purified from the milk of transgenic rabbits,
pigs, sheep, goats and cattle.

Therapeutic antibodies

Antibodies for human therapy are the fastest growing set of new biopharmaceuticals, in terms
of number of products and market share.

20 antibody products are approved for therapeutic use and several hundred are undergoing
trials for applications in cancer therapy, autoimmune diseases, transplantation, antibiotic
resistant infections, biodefense and immune deficiencies.

The first therapeutic monoclonal to gain regulatory approval was Ortho Biotechs orthoclone
OKT3 (Muromonab) antibody to T-cell CD3 antigen for the suppression of kidney transplant
rejection in June 1986.

Immunogenicity can be reduced by using proteolytic fragments containing the antigen

binding
regions (Fab fragments) rather than the whole mouse antibody.

Considerable efforts have been made to reduce the mouse content The first products were
chimeric antibodies composed of human constant regions fused with mouse variable regions.

A Fab fragment from a chimeric antibody was approved in 1994, Centocors Abciximab
(ReoPro) to block platelet aggregation after cardiac angioplasty or stent insertion.
The first whole chimeric antibody to be approved was from Biogen Idec and Genentech,

In 1994, both independently reported immunoglobulin knock-out mice carrying human

immunoglobulin transgenes. These mice could be immunised and hybridomas derived
producing fully human monoclonal antibodies

Polyclonal antibodies,

Therapeutic monoclonals, from whatever source, all share the feature that a single antibody
binds only one epitope of an antigen. In contrast, the normal vertebrate immune response
generates a population of antibodies with a range of affinities against various epitopes.

Monoclonals are therefore of limited effectiveness against pathogens which require

neutralisation of multiple epitopes, pathogens with diverse strains or which mutate rapidly,
and venoms with tens or hundreds of toxic components.

Polyclonals are also more effective than monoclonals in the formation of immune complexes
responsible for complement activation and the recruitment of neutrophils and macrophages.

Mice are not themselves a practical source of immune sera; each adult contains only about 10
mg total immunoglobulins.

But if the necessary genetic modifications can be performed, many larger species would be
eminently suitable; cattle have approximately 1 kg total immunoglobulins

Kirin Pharma have created transchromosomic cattle using a human artificial chromosome
vector containing the entire un-rearranged sequences of the human immunoglobulin G heavy-
chain and lambda light-chain loci

Herds of large animals capable of producing human polyclonal antibodies would transform
animal pharming from its current status as merely another means of producing pre-existing
proteins to a unique source of totally new pharmaceutical products.

Molly and Polly both carried the human gene that codes for a protein called Factor IX. Factor
IX is a blood clotting protein, and it is commonly used to treat patients that have hemophilia
B, a disease that is caused by a deficiency in Factor IX. The goal of creating Molly and Polly
was to produce a herd of sheep that produced Factor IX in their milk. The only other source
of Factor IX is from human blood plasma. By producing Factor IX in sheep milk, it is hoped
that the cost of production will be greatly reduced.

Advanced Cell Technology, Inc is using the queen of milk production, the cow, for potential
use as bioreactors. They have produced transgenic cows that secrete the protein serum
albumin in their milk, a protein that is used to extend blood volume and is used in patients
suffering from traumatic injuries, such as burns. Cows are an obvious choice for pharming
purposes as they can produce upwards of 8000 L of milk per year, and an estimated 40 to 80
kg of protein a year. That is quite a substantial amount compared to the 4 kg of protein per
year in goats and 2.5 kg of protein per year in sheep.
 Nexia Biotechnologies has been successful at producing spider silk in the milk of goats, and
is in the process of developing "Biosteel", a man made fabric made of spider silk. Potential
applications of this fabric are extensive, ranging from medical uses (wound closures,
dressing, patches, glues, prosthetic devices), to military uses (strong, light-weight body
armour), to sporting goods (biodegradable fishing line).
Xenotransplantation of porcine organs to human patients

Today .250 000 people are alive only because of the successful transplantation of an
appropriate human organ (allotransplantation). On average, 7590% of patients survive the
first year after transplantation and the average survival of a patient with a transplanted heart,
liver or kidney is 1015 years.

This progress in organ transplantation technology has led to an acute shortage of appropriate
organs, and cadaveric or live organ donation cannot cover the demand in western societies

porcine xenografts are considered the solution of choice because

(i) the organs are similar in size to human organs;
(ii) porcine anatomy and physiology are not too different from that of humans;
(iii) pigs have short reproduction cycles and large litters; (iv) pigs grow rapidly;
(v) maintenance of high hygienic standards is possible at relatively low costs; and
(vi) transgenic techniques for modifying the immunogenicity of porcine cells and organs
are well established.

The immunological obstacles in a porcine-to-human xenotransplantation are the hyperacute

rejection response (HAR), acute vascular rejection (AVR), cellular rejection and potentially
chronic rejection

When using a discordant donor species such as the pig, overcoming the HAR and AVR are
the preeminent goals. The most promising strategy for overcoming the HAR is the synthesis
of human complement regulatory proteins (RCAs) in transgenic pigs

Following transplantation, the porcine organ would produce the complement regulatory
protein and can thus prevent the complement attack of the recipient

Another promising strategy towards successful xenotransplantation is the knockout of the

antigenic structures on the surface of the porcine organ that cause HAR.

These structures are known as 1,3-a-gal-epitopes and are primarily produced by activity of
1,3-a-galactosyltransferase (a-gal). Piglets in which one allele of a-gal locus had been
knocked out by homologous recombination in primary donor cells that were employed in
nuclear transfer were recently generated

3. Industrial Applications

In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells
of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny
silk strands from their body by the bucketful. By extracting polymer strands from the milk
and weaving them into thread, the scientists can create a light, tough, flexible material that
could be used in such applications as military uniforms, medical microsutures, and tennis
racket strings.

Toxicity-sensitive transgenic animals have been produced for chemical safety testing.
Microorganisms have been engineered to produce a wide variety of proteins, which in turn
can produce enzymes that can speed up industrial chemical reactions.
Potential applications of this fabric are extensive, ranging from medical uses (wound
closures, dressing, patches, glues, prosthetic devices), to military uses (strong, light-weight
body armour), to sporting goods (biodegradable fishing line).

Gene therapy
Genetic engineering means that we alter an organism permanently so that the
changes will be stably inherited

In contrast, gene therapy (occasionally called genetic surgery) is less permanent.

The patient is cured, more or less, by altering the genes in only part of the body.
For example, cystic fibrosis patients might be partially cured by introducing the
wild-type gene into the lungs. However, these changes are not inherited

The most straightforward use of gene therapy is to deal with a hereditary defect
due to a single gene and that occurs only when both copies of the gene are
defective

Introducing a single good copy of the gene can then cure the defect. This is
sometimes known as replacement gene therapy

The main steps involved in replacement gene therapy are as follows:

(a) Identification and characterization of gene
(b) Cloning of gene
(c) Choice of vector
(d) Method of delivery
(e) Expression of gene

The first step is to identify the genetic defect and to clone a good copy of the
gene involved. The gene must then be delivered to the patient. This involves
choosing a vector together with a suitable method of delivery.

In addition, the vector/gene construct must be designed to allow proper

expression of the gene, once inside the patient.
Delivery may be performed in a variety of ways. The vector/gene construct may
be injected into the bloodstream or other tissue. It may be aerosolized and
sprayed into the nose and airways.

In some cases, cells are removed from the patient, engineered while growing in
culture, and then returned to the patient. This approach is known as ex vivo gene
therapy because the actual genetic engineering takes place outside the patient.

In most cases a modified virus is used as the vector. Because viruses cause
disease, they first need to be genetically disarmed in order to be used in gene
therapy. About 70% of human gene therapy trials have used viral vectors
Two main groups of viruses have been used, retroviruses and adenoviruses. In
addition, in a smaller proportion of cases DNA has been delivered inside
liposomes or projected into tissues by the gene gun

GENE PATCHING BY OLIGONUCLEOTIDE CROSSOVER

Some genetic defects consist of just a single base change, or perhaps a cluster of
closely linked base alterations. In this case, the defective gene could be
patched rather than replaced.

This may be done by crossing over with a relatively short double-stranded

oligonucleotide, which carries the wild-type sequence

The crossover frequency may be improved by using an RNA-DNA hybrid

oligonucleotide

AGGRESSIVE GENE THERAPY

We can go on the offensive and provide genes whose products may cure a
disease even though the genes we use were not responsible for the problem in
the first place.

The best examples of such aggressive gene therapy are not in curing hereditary
defects but in the treatment of cancer. Here the objective is to kill cancer cells

ADENOVIRUS VECTORS IN GENE THERAPY

Adenoviruses are relatively simple, double-stranded DNA viruses that infect

humans and other vertebrates. The virus particle consists of a simple icosahedral
shell, or capsid, containing a single linear dsDNA molecule of approximately
36,000 base pairs
Adenoviruses were among the first viruses chosen as vectors for use in human
gene therapy. Their advantages are as follows:

(a) Adenoviruses are relatively harmless. They cause mild infections of

epithelial cells, especially those lining the respiratory or gastrointestinal tracts.
(b) Adenoviruses are nononcogenic (i.e., they do not cause tumors).
(c) Adenoviruses are relatively easy to culture and can be produced in large
quantities.
(d) The life cycle and biology of adenovirus are well understood.
(e) The function of most adenovirus genes is known.
(f) The complete DNA sequence is available, in particular for adenovirus
serotype 5 of subgroup C.

Although mild, adenoviruses do cause inflammation and can cause serious

illness in patients with damaged immune systems.

Therefore, when designing an adenovirus vector for gene therapy, the virus
needs to be disarmed by crippling its replication system. This is done by
deleting the gene for E1A protein, a virus protein made immediately on
infection. E1A has two functions .

First, it promotes transcription of other early virus genes. Second, it binds to

host cell Rb protein, which normally prevents the cell from entering S-phase.

This prompts the host cell to express genes for DNA synthesis, which the virus
utilizes for its own replication.

Although genes carried on engineered adenovirus have been expressed

successfully in both animal and human tissues, there are problems.

The major difficulty is that adenovirus infections are short-lived. Thus the
therapeutic gene is expressed for only a few weeks before the immune system
eliminates the virus.

Furthermore, the patient develops immunity to the virus so that a second

treatment with the same engineered virus will fail. Thus adenovirus vectors
cannot be used for long-term gene therapy for hereditary diseases. Adenovirus
vectors may help deliver a deadly gene to cancer cells. Here only a short period
of expression should be needed

ADENO-ASSOCIATED VIRUS
Because of the problems with using adenovirus discussed above, other DNA
viruses have been considered as vectors. Although none are yet widely used, the
adeno-associated virus (AAV) shows considerable promise.

AAV is a defective or satellite virus that depends on adenovirus (or some

herpes viruses) to supply some necessary functions. Consequently, it is
usually found in cells that are infected with adenovirus.

Unlike adenovirus, AAV seems to be entirely harmless.

The benefits of using AAV are as follows:
(a) It does not stimulate inflammation in the host.
(b) It does not provoke antibody formation and can therefore be used for
multiple treatments.
(c) It infects a wide range of animals, as long as an appropriate helper virus is
also present. It can therefore be cultured in many types of animal cells,
including those from mice or monkeys.
(d) It can enter nondividing cells of many different tissues, unlike adenovirus.
(e) The unusually small size of the virus particle allows it to penetrate many
tissues of the body effectively.
(f) AAV integrates its DNA into a single site in the genome of animal cells (the
AAVS1 site on chromosome 19 in humans). This allows the therapeutic gene to
be permanently integrate

One drawback is that the AAV genome is small (4681 nucleotides of single-
stranded DNA) and the virus can carry only a relatively short segment of DNA.
(AAV is unusual in packaging both plus and minus strands into virus particles.

Although each virus particle contains only one ssDNA molecule, a virus
preparation contains a mixture of particles, half with plus and half with minus
strands.)

Mice and monkeys have been experimentally treated with a double AAV
system that provides erythropoietin, a protein required for development of red
blood cells.

RETROVIRUS GENE THERAPY

Retroviruses infect many types of cells in mammals. They need dividing cells
for successful infection, and will not infect many tissues where host cell growth
and division have come to a standstill.
Moreover, the genetic material of retroviruses passes through both DNA and
RNA stages. This means that introns must be removed from any therapeutic
genes before they are cloned into a retrovirus.

Despite these extra technical difficulties, a retrovirus has the distinction of

carrying the first gene in successful human gene therapy

Vectors for gene therapy have been derived from the simpler retroviruses,
especially murine leukemia virus (MuLV).

The vectors have all the retrovirus genes removed, and as a result they are
completely defective in replication. They retain only the packaging signal and
the two LTRs and can carry approximately 6 to 8 kb of inserted DNA. A virus
promoter in the 5 LTR drives expression of the cloned gene.

After infection of the patient, the RNA inside the retroviral vector is reverse
transcribed to give a DNA copy.

Because the retroviral vectors are completely devoid of genes for retrovirus
proteins, they do not cause an immune response or significant inflammation.

Furthermore, their ability to integrate into host cell DNA means that the
therapeutic gene will become a permanent part of the host cell genome.

More serious problems are that retroviral vectors can carry only small amounts
of DNA (about 8 kb) and cannot infect non dividing cells. However, the
lentivirus family of retroviruses (to which HIV belongs) is unusual in being able
to infect some nondividing cells.

Naturally, using HIV itself is risky, but a future possibility is to transfer this
ability into other, safer retroviruses.

The first successful instance of human gene therapy used a retroviral vector to
provide a functional copy of the Ada gene to a child with Severe combined
immunodeficiency SCID.

The cells affected by SCID are the lymphocytes that circulate in the blood,
where they carry out immune surveillance. They are produced by division of
bone marrow cells.

Gene therapy involves removing bone marrow cells from the patient and
maintaining them in cell culture outside the body. Because bone marrow cells
constantly replenish the blood supply, they divide often and are suitable for
retroviral infection.

While in culture, the bone marrow cells are infected with genetically engineered
retrovirus carrying the Ada gene and are then returned to the body.

Since 1991, several children have been treated by this approach.

NONVIRAL DELIVERY IN GENE THERAPY

However sophisticated a viral delivery system may be, nonviral vectors are
inherently safer. Nonetheless, they have been relatively neglected because
viruses were more efficient.

However, several unfortunate incidents have occurred with viral vectors,

especially retroviruses, including the occasional appearance of leukemia-like
disease.

This has resulted in renewed interest in nonviral delivery systems.

About 75% of gene therapy trials have used viral vectors. A variety of
alternative approaches have also been investigated, though few have been
effective or widely used so far.

These include:
(a) Use of naked nucleic acid (DNA or less often RNA). Many animal cells can
be transformed directly with purified DNA. The therapeutic gene may be
inserted into a plasmid and the plasmid DNA used directly. Some 10% to 20%
of gene therapy trials have used unprotected nucleic acid.
(b) Particle bombardment. DNA is fired through the cell walls and membranes
on metal particles. This method was originally developed to get DNA into
plants. However, it has also been used to make transgenic animals and is
occasionally used for humans.
(c) Receptor-mediated uptake. DNA is attached to a protein that is recognized
by a cell surface receptor. When the protein enters the cell, the DNA is taken in
with it.
(d) Polymer-complexed DNA. Binding to a positively charged polymer, such as
polyethyleneimine, protects the negatively charged DNA. Such complexes are
often taken up by cells in culture and may in principle be used for ex vivo gene
therapy.
(e) Encapsulated cells. Whole cells engineered to express and secrete a needed
protein may be encapsulated in a porous polymeric coat and injected locally.
Foreign cells excreting nerve growth factor have been injected into the brains of
aging rats. The rats showed some improvement in cognitive ability, suggesting
that this approach may be of value in treating conditions such as Alzheimers
disease.
(f) Liposomes are spherical vesicles composed of phospholipid. They have been
used in around 10% of gene therapy trials
Antisense RNA
RNA plays a multifaceted role in biology that is adaptable for many different
applications in biotechnology. It is now known that important gene regulation
occurs at the level of RNA.

In some organisms, antisense RNA is one way by which RNA controls protein
translation. Antisense RNA binds to the complementary mRNA and blocks
translation.

From this discovery came the potential use of antisense RNA to block or
attenuate synthesis of proteins that cause various diseases.

A second method of RNA-regulated gene expression is RNA interference

(RNAi). Here small noncoding RNAs identify specific mRNAs and trigger their
degradation.

The two complementary strands of DNA are referred to as sense (= coding or

plus) and antisense (= noncoding or minus).

Transcription occurs using the antisense strand as template, resulting in an

mRNA that is identical in sequence to the sense strand (except for the
replacement of uracil for thymine).

Antisense RNA is synthesized using the sense strand as template; therefore, it

has a sequence complementary to mRNA

When a cell has both the mRNA (i.e., the sense strand of RNA) plus a
complementary antisense copy, the two single strands anneal to form double-
stranded RNA. The duplex can either inhibit protein translation by blocking the
ribosome binding site, or inhibit mRNA splicing by blocking a splice site

RNase H is a cellular enzyme that normally functions during replication. It

recognizes and cleaves the RNA backbone of an DNA:RNA duplex, targeting
the antisense DNA:mRNA duplex for further degradation.
RNase H recognizes a 7-base-pair heteroduplex, so the region of homology
between the antisense DNA and target mRNA need not be very long.

In the laboratory, antisense RNA can be made by two different methods. The
easiest method is to chemically synthesize oligonucleotides that are
complementary to the target gene.

The oligonucleotides are then injected or transformed into the target cell.

Alternatively, the gene of interest can be cloned in the opposite orientation, so

that transcription gives antisense RNA. The vector carrying the anti-gene is
then transformed into the target organism.
RNA interference uses antisense sequences to inhibit Gene expression

RNA interference (RNAi) is a recently discovered pathway for endogenous

gene regulation where short double-stranded RNA (dsRNA) segments trigger an
enzyme complex to degrade a target mRNA.

In essence, the short dsRNA pieces decrease target protein expression

by degrading its complementary mRNA.

RNAi was discovered in a variety of different organisms, including plants,

fungi, mammals, flies, and worms, and consequently has multiple names.

Different organisms have variations of the same basic response. Mutations

in the enzymes responsible for RNAi affect a wide range of cellular processes.
Some affect development of the organism; others affect the ability to fend off
viruses, particularly RNA viruses
RNAi is divided into two different phases, the initiation phase and the effector
phase.

Initiation begins by the formation of dsRNA. The dsRNA can arise from three
main sources.

1. infecting RNA viruses replicate through a dsRNA intermediate, which can

trigger RNAi.

2. the organisms own genomic DNA contains sequences that code for
microRNAs, which are specific mediators of RNAi

3.dsRNA can be produced from aberrant transcription of a genetically

engineered gene

During the next step of initiation, dsRNA binds to an endonuclease called Dicer,
which cuts the dsRNA into small fragments about 21 to 23 nucleotides in length
called short interfering RNAs.

Dicer is a dsRNA-dependent RNA endonuclease that belongs to the RNase III

family.

The siRNAs have a two-nucleotide overhang on the 3 ends (characteristic of

RNase IIItype enzymes). The 5 ends are phosphorylated by a kinase
associated with Dicer, making the siRNAs competent for the next phase

In the effector phase, Dicer transfers the siRNA to a ribonucleoprotein complex

called the RNA-induced silencing complex (RISC).

RISC is activated by the siRNAs and uses an RNA helicase to unwind the
double-stranded fragments, making single strands.

The antisense single-stranded siRNA is then kept as a guide to find

complementary sequences in the cytoplasm.

When RISC binds complementary sequences, these are first cleaved by an

endonuclease within the RISC complex and are then further degraded by
exonucleases in the cytosol.

This destroys all of the mRNA that is complementary to the siRNA.

Both Dicer and the RISC complex are dependent on ATP for energy.
The antisense siRNA specifies which mRNA is targeted, ensuring that no
nonspecific mRNAs are degraded.

The ability to target so many mRNA molecules with so few siRNA copies relies
on amplification by the enzyme RNA-dependent RNA polymerase (RdRP),
which creates dsRNA.

RdRP uses the cleaved target mRNA as template to synthesize more dsRNA.

Dicer recognizes the new dsRNA and cleaves it into more siRNA, thus greatly
increasing the number of siRNA molecules
Characteristics of RNAi

RNAi is highly efficient in restraining the expression of the target gene.

The target mRNA often starts to degenerate after 46 h when siRNA enters the
cell

siRNA combines strictly with the target mRNA according to the base pairing
principle. It requires quite high specificity of complementary sequences

If one base difference with the sequence of complementary mRNA exists, the
potency with which the siRNA restrains the expression of the target gene will
decline dramatically and for two or three base differences, the siRNA function
will be completely lost.

Application of RNAi in plants

Application for functional genomics

RNAi has been utilized, for some time, for plant functional genomics with its
interruption of gene expression.

dsRNA triggers the degradation of homologous RNAs, thus preventing gene

expression.

The primary advantage is an ability to target specifically a chosen gene. The

selection of a unique region of the target sequence can ensure that a specific
gene family member is silenced, or multiple members of a gene family can be
silenced by targeting highly conservative sequence domains.

RNAi can also be used to analyze the functions of essential genes. Variable
levels of gene silencing can be achieved in different transgenic lines using the
same ihpRNA (hairpin RNA which contains an intron) construct.

At present, there are two ways to trigger RNAi in plants. One is an expression
vector used for plant genetic transformation such as the Agrobacterium strain
in which T-DNA regains the inserted DNA fragment for ihpRNA induction.

The other way is virus-induced gene silencing (VIGS), in which the target
sequence is integrated into viral sequences that are used to infect the plant, or
expressed from Agrobacterium- introduced transgenes and by stable
transformation with ihpRNA expressing transgenes.

So far, many novel gene functions have been detected by RNAi.

Application in genetic improvement of plants
RNAi technology is a kind of highly efficient knockdown technology in plants
and it provides a useful way for genetic improvement

The rice mutant line LGC-1 (Low Glutelin Content-1) was the first
commercially useful cultivar produced by RNAi.

This low-protein rice is useful for patients with kidney disease whose protein
intake is restricted. leading to lower the storage protein glutelin content in rice

Inhibited the theobromine synzyme gene by RNAi and decreased caffeine by

70% of the normal level.

Modified fatty acid composition of cottonseed oil using the technology of

hpRNA-mediated gene silencing to down-regulate the seed expression of two
key fatty acid desaturase genes, ghSAD-1 which encodes stearoyl-acyl-carrier
protein 9-desaturase and ghFAD2-1 which encodes oleoyl-
phosphatidylcholine 6-desaturase

silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content,
up to 77% compared with about 15% in seeds of untransformed plants.

Similar fatty acid composition phenotypes were also achieved

RNAi shows that it has an advantage in that it can improve its applicability to
multigene families and polyploids (Lawrence and Pikaard, 2003), since it is not
straightforward to knock out a multigene family by the accumulation of
mutations for each member of the family by conventional breeding,

Application as antivirus defense in plants

Most known plant viruses have RNA genomes and replicate via dsRNA
intermediates, thereby serving as potent inducers of RNAi during early
replication and as silencing targets in later infections.

In an attempt to protect barley against BYDV, Golden Promise barley was

transformed with a DNA segment that was designed to produce hairpin RNA
from BYDV-PAV sequences.

Missiou and his group (2004) applied RNAi to engineer potato plants that are
resistant to the potato virus Y (PVY). They expressed dsRNA derived from the
3 terminal part of the coat protein gene of PVY, which is highly conserved in
sequence amongst different PVY isolates, in transgenic potatoes of the
commercial variety Spunta.

Applications in Animals

Antisense therapy is a form of treatment for genetic disorders or infections.

When the genetic sequence of a particular gene is known to be causative of a
particular disease, it is possible to synthesize a strand of nucleic acid
(DNA, RNA or a chemical analogue) that will bind to the messenger
RNA (mRNA) produced by that gene and inactivate it, effectively turning that
gene "off"

Hemorrhagic fever viruses, Cancer, HIV/AIDS, High cholesterol

Unit V PROTEIN PRODUCYION
Insulin production
The nature and purpose of synthesising human insulin.

Since Banting and Best discovered the hormone, insulin in

1921.(1) diabetic patients, whose elevated sugar levels (see fig. 1) are
due to impaired insulin production, have been treated with insulin
derived from the pancreas glands of abattoir animals. The hormone,
produced and secreted by the beta cells of the pancreas' islets of
Langerhans,(2)regulates the use and storage of food, particularly
carbohydrates.

Although bovine and porcine insulin are similar to human insulin, their
composition is slightly different. Consequently, a number of patients'
immune systems produce antibodies against it, neutralising its actions
and resulting in inflammatory responses at injection sites. Added to
these adverse effects of bovine and porcine insulin, were fears of
long term complications ensuing from the regular injection of a
foreign substance,(3) as well as a projected decline in the production
of animal derived insulin.(4) These factors led researchers to consider
synthesising Humulin by inserting the insulin gene into a suitable
vector, the E. coli bacterial cell, to produce an insulin that is
chemically identical to its naturally produced counterpart. This has
been achieved using Recombinant DNA technology. This method
(see fig. 2) is a more reliable and sustainable(5) method than
extracting and purifying the abattoir by-product.

The structure of insulin.

Chemically, insulin is a small, simple protein. It consists of 51 amino

acid, 30 of which constitute one polypeptide chain, and 21 of which
comprise a second chain. The two chains (see fig. 3) are linked by a
disulfide bond
Biological implications of genetically engineered Recombinant
human insulin.

Human insulin is the only animal protein to have been made in

bacteria in such a way that its structure is absolutely identical to that
of the natural molecule. This reduces the possibility of complications
resulting from antibody production. In chemical and
pharmacological studies, commercially available Recombinant DNA
human insulin has proven indistinguishable from pancreatic human
insulin.(12) Initially the major difficulty encountered was the
contamination of the final product by the host cells, increasing the
risk of contamination in the fermentation broth. This danger was
eradicated by the introduction of purification processes. When the
final insulin product is subjected to a battery of tests, including the
finest radio-immuno assay techniques,(13) no impurities can be
detected.(14) The entire procedure is now performed using yeast cells
as a growth medium, as they secrete an almost complete human
insulin molecule with perfect three dimensional structure. This
minimises the need for complex and costly purification procedures.

Human growth hormone was, after insulin, the second product of this new
technology. This product was developed and commercialized initially by

Genentically engineered GH could be used for treating growth problems and

dwarfism (1). Furthermore, growth hormones from different animal species
have also been produced in transgenic organisms and these have been used in
different examples in the aquatic animal and livestock sectors

The biotechnological potential of GHs could be enormous, since besides its use
in species of the same origin, it has been demonstrated that the GHs of
mammals have activity in phylogenetically lower animals.

For example, BGH and porcine GH (PGH) have been used experimentally for
the treatment of hypophyseal dwarfism in dogs and cats .

Since the recombinant protein is frequently recovered from E. coli with

undesirable modifications (extra methionine, incorrect folding, aggregated
forms, etc.) and contaminated with highly pyrogenic substances, toilsome
purification schemes are needed to obtain it with the desired purity, structure
and biological activity.

For this, subsequent efforts have focused on the search for better expression
systems, with production being attempted with Saccharomyces cerevisiae (39),
Bacillus subtilis mammal cell cultures as well as transgenic animals

Unfortunately, these expression systems do not offer a production level greater

than that of E. coli and therefore in most cases they are not profitable

Yeasts offer the best of both prokaryotes and eukaryotes, since, in addition to
performing some of the post-translational modifications that are common in
superior organisms, they are easily grown in flasks and bioreactors, like
bacteria, using simple and inexpensive culture media

Obtain, clone, and manipulate cDNA from these hormones.

b. Construct and insert into the genome of P. pastoris the hormones expression
cassettes.
c. Develop the fermentation processes for each new strain.
d. Implement the purification schemes of the recombinant hormones.
e. Evaluate in vitro the bioactivity of the semipurified recombinant hormones..
 Biodegradable Plastics

Conventional polymers such as polyethylene and polypropylene persist for

many years after disposal. Built for the long haul, these polymers seem
inappropriate for applications in which plastics are used for short time
periods and then disposed.

Furthermore, plastics are often soiled by food and other biological substances,
making physical recycling of these materials impractical and generally
undesirable.

In contrast, biodegradable polymers (BPs) disposed in bioactive environments

degrade by the enzymatic action of microorganisms such as bacteria, fungi, and
algae. Their polymer chains may also be broken down by non enzymatic
processes such as chemical hydrolysis.

BPs are often derived from plant processing of atmospheric CO2.

Biodegradation converts them to CO2, CH4 water, biomass, humic matter, and
other natural substances. BPs are thus naturally recycled by biological processes

Biodegradable Plastics from Starchand Cellulose

Starch is an inexpensive, annually renewable material derived from corn and
other crops.

The biodegradation of starch products recycles atmospheric CO2 trapped by

starch-producing plants. All starches contain amylose and amylopectin, at ratios
that vary with the starch source.

This variation provides a natural mechanism for regulating starch material

properties. Starch-based BPs can be produced by blending or mixing them with
synthetic polymers.

By varying the synthetic blend component and its miscibility with starch, the
morphology and hence the properties can be regulated easily and efficiently.

This approach has been successfully implemented by Novamont under the

Mater-Bi trademark. Blends containing thermoplastic starch (destructurized
starch that is noncrystalline, produced by the application of heat and work) may
be blended or grafted with biodegradable polyesters [such as polycaprolactone
(PCL)] to increase flexibility and resistance to moisture.
Eastman Chemical Company has developed fully biodegradable cellulose
acetates that are promising but not yet commercially available

Biodegradable Plastics from Polyesters

Polyhydroxylalkanoates (PHAs) are produced directly from renewable

resources by microbes. They can be accumulated to high levels in bacteria
(_95% of the cellular dry weight), and their structures can be manipulated
by genetic or physiological strategies.

The physical properties and biodegradability of PHAs can be regulated by

blending with synthetic or natural polymers.

Sommerville developed genetically altered plants that contained

the necessary metabolic pathway to accumulate PHAs.

In an attempt to bring this technology to market, Monsanto spliced the plastic-

producing gene sequence into canola plants.

They created a plant that was not only 14% plastic, but also could be used
to produce canola oil. However, Monsanto believed that PHA-producing plants
would not be commercially viable unless 20% or more of the plant was plastic.

Metabolix (Cambridge, MA) continues to pursue the commercialization of

PHAs both in plant crops and by fermentation processes

Recent efforts to express the copolymer in Escherichia coli also hold promise
for reducing production cost and simplifying purification.

Poly(lactic acid) (PLA)

The manufacture of polyester from lactic acid was pioneered by Carothers in

1932 and further developed by Dupont and Ethicon.

Prohibitive production costs restricted the applicability of these polymers

outside the medical field until the late 1980s. Since then, major breakthroughs
in process technology, coupled with decreased costs of biologically produced
lactic acid, have led to the commercial-scale production of BPs from lactic acid
for nonmedical applications.
This integration of biotechnology and chemistry is an important strategy that
will be critical toimprovements in many other chemical processes in future
years.

PLA is currently used in packaging (film, thermoformed containers, and short

shelflife bottles).

Cargill Dow LLC uses conventional melt-spinning processes to form fibers

for clothing and other uses. Fabrics produced from PLA provide a silky feel,
durability, and moisture-management properties (moisture is quickly wicked
away from the body, keeping the wearer dry and comfortable).

Poly(-caprolactone), PCL, and poly(alkyene succinate)s. PCL is a

thermoplastic biodegradable polyester synthesized by chemical conversion of
crude oil, followed by ring-opening polymerization.

Triparental mating
Triparental mating is a form of Bacterial conjugation where a conjugative
plasmid present in one bacterial strain assists the transfer of a mobilizable
plasmid present in a second bacterial strain into a third bacterial strain.

Plasmids are introduced into bacteria for such purposes

as transformation, cloning, or transposon mutagenesis. Triparental matings can
help overcome some of the barriers to efficient plasmid mobilization. For
instance, if the conjugative plasmid and the mobilizable plasmid are members of
the same incompatibility group they do not need to stably coexist in the second
bacterial strain for the mobilizable plasmid to be transferred.

Requirements

A helper strain, Carrying a conjugative plasmid (such as the F-plasmid) that

codes for genes required for conjugation and DNA transfer.
A donor strain, Carrying a mobilizable plasmid that can utilize the transfer
functions of the conjugative plasmid.
A recipient strain, you wish to introduce the mobilizable plasmid into.

Five to seven days are required to determine if the plasmid was successfully
introduced into the new bacterial strain and confirm that there is no carryover of
the helper or donor strain.
In contrast, Electroporation does not require a helper or donor strain. This helps
avoid possible contamination with other strains. The introduction of the plasmid
can be verified in the recipient strain in two days, making electroporation a
faster and more efficient method of transformation.

nIndustrial Applications of Recombinant DNA

Technology
Proteins for industrial applications include those used as purification and
diagnostic tools as well as enzymes for industrial processes that may be as
diverse as food processing and paper and pulp bleaching.

Enzymes were initially obtained as natural products from animal, plant and/or
microbial tissues. Originally, the largest number of such enzymes were obtained
from plant and animal sources, but as microbial fermentation became more cost-
effective, this system provided the bulk of commercial enzymes from natural
sources.
However, natural sources often have disadvantages as source material for
enzyme production for a variety of reasons including limitations in the amount
of that material, geographical availability of that material, and cost of the
material.

Therefore, foreign protein production systems were developed as sources of

important industrial products. Xenogenic protein production has been
accomplished in bacteria, fungi, cultured animal cells, transgenic animals and of
highest interest here, plants .

Bacteria and fungi are relatively simple systems; however, these microbial
systems require a large capital outlay initially for fermentation equipment.
Bacteria may efficiently synthesize and secrete proteins and enzymes that are
not glycosylated.

In many cases, this is acceptable. However, if glycosylation is required, the

bacterial system is not appropriate. The bacterial-sourced proteins may be easily
purified if they remain soluble.

Fungi produce glycoproteins that can be secreted and are relatively easy to
purify. However, in some cases, mis-folding of proteins in bacteria and hyper-
glycosylation of proteins in fungi may occur, making alternative production
systems necessary.

Animal cell culture and transgenic animal production of foreign proteins are
currently being explored in academic and industrial laboratories. The major
advantage of these systems is that the sugars at the glycosylation sites more
nearly resemble those of the native protein in animals.

These systems are expensive and increasing the size of the herd of transgenic
animals is quite slow. Therefore, transgenic animals and cells are most often
only cost effective for high value proteins.

The advantages of plant production systems

Transgenic plants provide a viable technology for producing protein products

They have many advantages including low cost of production, stability of

protein products in storage tissues such as seeds, ease and speed of scale-up and
the possibility of direct addition of plant material to industrial processes.
In addition, plant systems have expressed proteins with integrity across a wide
range of conformations and molecular weights, including proteins such as
trypsin and a laccase isozyme that were not successfully expressed in
fermentation systems.

The production cost of enzymes from any source includes several factors such
as raw materials, processing and possibly purification. For an enzyme that
requires purification, the raw material can be a minor part of the overall cost of
the productin some cases as little as 10% depending on purity.

If a plant extract and fungal fermentation broth offer a protein at the same
concentration, then no difference is seen in the final cost of production.
However, if an extract is 10 fold more concentrated, the cost advantage for a
partially purified product is significantly better, because processing costs are
directly related to concentration of the starting material.

The advantage that plants have in this regard is that seed can offer proteins at a
much higher concentration than fermentation broth in the initial extract because
of the expression levels that can be achieved.

This will not be true for all enzymes, of course, but particularly for those
enzymes that do not express or are poorly expressed in fermentation cultures.
Plant production of proteins involves many steps.

The gene itself may require engineering so that the signals are recognized by the
plant transcription and translation machinery. The signals required include a
promoter, a subcellular targeting signal, and a terminator. The vector of choice
depends on the transformation protocol being employed.

New promoters are being isolated from all plant systems being utilized today.
These promoters take advantage of tissue-type specificity as well as preferred
sinks in the plant or seed.

Examples of industrial proteins from transgenic plants

ProdiGene has expressed and produced four industrial proteins in transgenic

maize- Native E. coli GUS, Maize-derived GUS, Egg white avidin, Maize-
derived avidin. The recombinant proteins are functionally equivalent to the
protein from native sources

Laccase is a blue copper oxidase with applications in the wood products and
textile industries, depending on enzyme properties.
 The gene for the laccase 1 isozyme from Trametes versicolor has been
expressed in transgenic maize.

Protease production in transgenic plants is best accomplished through zymogen

production, preferably in a seedspecific manner. This concept is protected by
U.S. Patent #6,087,558. Using this method, ProdiGene has produced
transgenic lines in maize that express trypsin at high levels of trypsin

Some plant-made industrial proteins/enzymes that have received considerable

interest - most of them are hydrolases, including glycosidases (e.g., cellulase, -
amylase

Zeigler recently reported expression of another industrial enzyme in a

transgenic plant. A themostable bacterial cellulase (endo-1,4-p-I-glucanase)
was produced in Arabidopsis thaliana.

Though a cell-wall degrading enzyme would be a priori a bad idea to produce

in a plant, this high temperature optimum (81C) enzyme is not active in tissues
at normal growth temperatures.

Therefore, even though the enzyme was secreted into the cell wall of the
transgenic plants, and thus is in direct contact with its target substrate, no
detrimental effects on the plants were detected. Although this enzyme
accumulated to as much as 25%

Another cell wall degrading enzyme , -amylase expressed in plants is a fungal

xylanase in canola. This enzyme was fused to an oleosin protein and so
immobilized on oil bodies; the oil body system allows recycling of enzyme for
reuse. Although its activity was similar to soluble enzyme produced in E. co&
expression levels are too low for commercial production (300-2000 units/kg
seed).

Development of Microbial recombinant production strains

Industrial production of recombinant enzymes is preceded by an extensive

research and development phase that culminates in the construction of a
successful production strain.

This process typically involves the following stages:

(1) development of the host (recipient) strain; (2) construction of the expression
vector; (3) transformation of the host strain; (4) identiWcation of the best
recombinant strain; (5) additional improvements; and (6) characterization of the
production strain.
Subtilisin production
Subtilisin (serine endopeptidase) is a non-specific protease (a protein-
digesting enzyme) initially obtained from Bacillus subtilis.
Subtilisins belong to subtilases, a group of serine proteases that initiate
the nucleophilic attack on the peptide (amide) bond through a serine residue at
the active site. They are physically and chemically well-characterized enzymes.
Subtilisins typically have molecular weights of about 20,000 to 45,000 dalton.
They can be obtained from soil bacteria, for example, Bacillus
amyloliquefaciens. Subtilisins are secreted in large amounts from
many Bacillus species.
Subtilisins are widely used in commercial products, for example, in laundry and
dishwashing detergents, cosmetics, food processing, skin care ointments,
contact lens cleaners, and for research purposes in synthetic organic chemistry.
Subtilisins are of bacterial origin, and are produced by a fermentation process.
The total amount of Subtilisin produced and used in the European Union in
2002 was about 1,000 tons of pure enzyme.

Nowadays usually genetically modified (engineered) microorganism (GMM)

strains are used, to enhance productivity. Sometimes these strains are also
producing protein-engineered Subtilisin variants to introduce a desired trait,
such as enhanced oxidative stability.
Some of the important enzymes are listed below

Safety Guideline for Recombinant DNA and Disposal

The new capabilities to manipulate the genetic material present tremendous potential and find use in many novel
experiments and applications. These developments have generated a sense of concern among scientists working in biological
areas and others to find ways how safely the research in the field should be carried out and means to regulate work involving
pathogenic microorganisms and genes of virulence.
A large number of research institutions in Government, Universities and private R&D labs have active biotech programmes
where research is being done in both in basic and applied fronts utilising microorganisms plant and animals, tissue
culture and cell lines and on development of vaccines towards communicable diseases of both men and animals. A good deal
of effort is being made in the areas of diagnostics, biofertilizers, biocides, fertility control, tissue culture of high value crops
to develop technologies and useful products.

The guidelines cover areas of research involving genetically engineered organism. It also deals with genetic transformation
of green plants, rDNA technology in vaccine development and on large scale production and dekliberate/ accidental release
of organisms, plants, animals and products derived by rDNA technology into the environment.

The guidelines are in respect of safety measures for the research activities, large scale use and also the environmental impact
during field applications of genetically altered material products

Research: The levels of the risk and the classification of the organisms within these levels based on pathogenicity and local
prevalence of diseases and on epidemic causing strains Some of the microorganisms not native to the country have been
assigned to a special category requiring highest degree of safety.
The guidelines employ the concept of physical and biological containment and also based upon the principle of good
laboratory practice (GLP).

Large scale operations: The concern does not diminish when it comes to the use of recombinant organisms scale
fermentation operations on large scale fermentation operations or applications of it in the environment. As such, the
guidelines prescribe criteria for good large scale practices (GLSP) for using recombinant organisms. These include measures
such as proper engineering for containment, quality control, personnel protection, medical surveillance, etc.

Environmental risks: Application and release of engineered organisms into the environment could lead to ecological
consequences and potential risks unless necessary safeguards are taken into account. The guidelines prescribe the criteria for
assessment of the ecological aspects on a case by case basis for
planned introduction of rDNA organism into the environment. It also suggests regulatory measures to ensure safety for
import of genetically engineered materials, plants and animals. The recommendations also cover the various quality control
methods needed to establish the safety, purity and efficacy of
rDNA products.

GUIDELINES

Classification of a pathogenic microorganisms

The classification of infective microorganisms are drawn up under 4 risk groups in increasing order of risk
Based on the risk assessment information, the probability of risk could be further assigned certain quantitative values
Containment
Containment facilities for different Risk Groups as per the recommendations of World Health Organization (WHO)
The term "Containment" is used in describing the safe methods for managing infectious agents in the laboratory environment
where they are being handled or maintained.

Purpose of containment
To reduce exposure of laboratory workers, other persons, and outside environment to potentially hazardous agents.
Types of containment

Biological containment (BC):

In consideration of biological containment, the vector (plasmid, organelle, or virus) for the recombinant DNA and the host
(bacterial, plant, or animal cell) in which the vector is propagated in the laboratory will be considered together.

Any combination of vector and host which is to provide biological containment must be chosen or constructed to limit the
infectivity of vector to specific hosts and control the host-vector survival in the environment.

These have been categorized into two levels - one permitting standard biological containment and the other even higher that
relates to normal and disabled host-vector systems respectively

Physical Containment (PC):

The objective of physical containment is to confine recombinant organisms thereby preventing the exposure of the
researcher and the environment to the harmful agents. Physical containment is achieved through the use of
i) Laboratory Practice,
ii) Containment
Equipment, and
iii) Special Laboratory Design.
 The protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by
good microbiological techniques and the use of appropriate safety equipment, (Primary Containment).
The protection of the environment external to the laboratory from exposure to infectious materials, is provided by a
combination of facility design and operational practices, (Secondary Containment).

Elements of Containment:

The three elements of containment include laboratory practice and

technique, safety equipment and facility design.
i) Laboratory practice and technique:
Strict adherence to standard microbiological practices and techniques
Awareness of potential hazards
Providing/arranging for appropriate training of personnel
Selection of safety practices in addition to standard laboratory practices if required
Developing of adopting a biosafety or operations manual which identifies the hazards

ii) Safety equipment (primary barriers): Safety equipment includes biological safety cabinets and a variety of enclosed
containers (e.g. safety centrifuge cup). The biological safety cabinet (BSC) is the principal device used to provide
containment of infectious aerosols generated by many microbiological procedures. Three types of BSCs (Class I, II, III) are
used in microbiological laboratories. Safety equipment also includes items for personal protection
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields and safety glasses,
etc.
iii) Facility Design (Secondary barriers): The design of the facility is important in providing a barrier to protect persons
working in the facility but outside of the laboratory and those in the community from infectious agents which may be
accidentally released from the laboratory.
There are three types of facility designs: viz, the Basic Laboratory (for Risk Group I and II), the Containment Laboratory
(for Risk Group III) and the Maximum Containment Laboratory
(for Risk Group IV).

MECHANISM OF IMPLEMENTATION OF BIOSAFETY GUIDELINES

For implementation of the guidelines it is necessary to have an institutional mechanism to ensure the compliance of requisite safeguards at
various levels. The guidelines prescribe specific actions that include establishing safety procedures for rDNA research, production and
release to the environment and setting up containment conditions for certain experiments. The guidelines suggest compliance of the
safeguards through voluntary as well as regulatory approach. In this connection, it is proposed to have a mechanism of advisory and
regulatory bodies to deal with the specific and discretionary actions on the following:
a. Self regulation and control in the form of guidelines on recombinant research activities
b. Regulation of large scale use of engineered organisms in production activity and release of organisms in
environmental applications under statutory provisions. The institutional mechanism as proposed for implementation of guidelines is shown
in organogram in Figure 2.
Mainly it consists of the following:-
i) Recombinant DNA Advisory Committee (RDAC)
ii) Institutional Biosafety Committee (IBSC)
iii) Review Committee on Genetic Manipulation (RCGM)
iv) Genetic Engineering Approval Committee (GEAC)

Recombinant DNA Advisory Committee (RDAC):

The Committee should take note of developments at national and international levels in Biotechnology towards the currentness of the safety
regulation for India on recombinant research use and applications. It would meet once in 6
months or sooner for this purpose.
The specific terms of reference for Recombinant Advisory Committee include the following :
i) To evolve long term policy for research and development in Recombinant DNA research.
ii) To formulate the safety guidelines for Recombinant DNA Research to be followed in India.
iii) To recommended type of training programme for technicians and research fellows for making
them adequately aware of hazards and risks involved in recombinant DNA research and methods of avoiding it.
Institutional Biosafety Committee (IBSC)
Institutional Biosafety Committee (IBSC) are to be constituted in all centres engaged in genetic engineering research and
production activities. The Committee will constitute the following:
(i) Head of the Institution or nominee
(ii) 3 or more scientists engaged in DNA work or molecular biology with an outside expert in the relevant discipline.
(iii) A member with medical qualifications - Biosafety Officer (in case of work with pathogenic agents/large scale use).
(iv) One member nominated by DBT.
The Institutional Biosafety Committee shall be the nodal point for interaction within institution for implementation of the
guidelines. Any research project which is likely to have biohazard potential (as envisaged by the guidelines) during the
execution stage or which involve the production of either
microorganisms or biologically active molecules that might cause bio-hazard should be notified to IBSC. IBSC will allow
genetic engineering activity on classified organisms only at places where such work should be performed as per guidelines.
Provision of suitable safe storage facility of donor, vectors, recipients and other materials involved in experimental work
should be made and may be subjected to inspection on accountability.

Review Committee on Genetic Manipulation (RCGM):

The RCGM will have the following composition:
i) Department of Biotechnology
ii) Indian Council of Medical Research
iii) Indian Council of Agricultural Research
iv) Council of Scientific & Industrial Research
v) Three Experts in Individual capacity
vi) Department of Science & Technology
The RCGM will have the functions:
i) To establish procedural guidance manual - procedure for regulatory process with respect to activity involving genetically
engineered organisms in research, production and applications related to environmental safety.
ii) To review the reports in all approved ongoing research projects involving high risk category and controlled field
experiments, to ensure that safeguards are maintained as per guidelines.
iii) To recommended the type of containment facility and the special containment conditions to be followed for experimental
trials and for certain experiments.
iv) To advise customs authorities on import of biologically active material, genetically engineered substances or products
and on excisable items to Central Revenue and Excise.
v) To assist Department of Industrial Development, Banks towards clearance of applications in setting up industries based on
genetically engineered organisms.
vi) To assist the Bureau of Indian Standards to evolve standards for biologics produced by rDNA technology.
vii) To advise on intellectual property rights with respect to rDNA technology on patents

Genetic Engineering Approval Committee (GEAC):

Genetic Engineering Approval Committee (GEAC) will function under the Department of Environment (DOEn) as statutory
body for review and approval of activities involving large scale use of genetically engineered organisms and their products
in research and development, industrial production, environmental release and field applications.
The functions include giving approval from environmental angle on:
i) Import, export, transport, manufacture, process, selling of any microorganisms or genetically engineered substances or
cells including food stuffs and additives that contains products derived by Gene Therapy.
ii) Discharge of Genetically engineered/classified organisms/cells from Laboratory, hospitals and related areas into
environment.
iii) Large scale use of genetically engineered organisms/classified microorganisms in industrial production and applications.
(Production shall not be commenced without approval).
iv) Deliberate release of genetically engineered organisms. The approval will be for a period of 4 years.
Institutional mechanism for implementation of guidelines frame work for implementation

GOI - Government of India, DBT - Department of Biotechnology

RDAC - Recombinant DNA Advisory Committee, IBSC - Institutional Biosafety Committee
RCGM - Review Committee on Genetic Manipulation, DOEn - Department of Environment
GEAC - Genetic Engineering Approval Committee SBCC - State Biotechnology Coordination Committee, PI - Principal Invstigator (R&D/Industry/Others)
FA - Funding Agency (Govt./Private & Public Institutions

Safety Precautions for Laboratories Using recombinant DNA (rDNA)

Laboratory Access
Only authorized persons may enter the laboratory. An authorized person must understand the safety precautions of a
recombinant DNA laboratory or be accompanied by one who does. Laboratory doors should be kept closed and locked
except when a laboratory worker is present. All entrances to the laboratory from the hallway should be marked with contact
information regarding the faculty member(s) in charge for the use of emergency personnel
Protective clothing
Goggles or safety glasses should be worn in the laboratory by all personnel at all times.
Closed Toe Footware should be worn in the laboratory by all personnel at all times. No sandals, flip-flops, etc.
Laboratory coats should be worn in the laboratory whenever rDNA work is being done. Do not wear these coats into areas
in which food or drink may be consumed. Isolate them in a plastic bag when taking them to the laundry.
Disposable gloves must be worn when handling microorganisms or nucleic acids. Change gloves immediately after obvious
contamination or tears. Do not leave the laboratory and handle door-knobs, etc. while still wearing gloves. Wash your hands
before leaving.
Goggles or safety glasses with a UV-protective coating are required whenever gels are observed on an ultraviolet
transilluminator. Both goggles AND a face shield are required for longer exposures, such as cutting out bands. Eye damage
and serious facial sunburn may result if these precautions are ignored.
Masks should be worn when weighing out or cleaning up dangerous powders such as ethidium bromide, acrylamide and
SDS. Always read labels before handling chemicals.
Laboratory behavior
Food, drink, gum-chewing, smoking, and application of cosmetics are prohibited in the laboratory.
No food or drink may be stored in refrigerators or elsewhere in the laboratory.
Mouth pipetting is prohibited.
Work Surfaces and Spills
Bench surfaces and microcentrifuges must be disinfected with a suitable agent (such as 70% ethanol, Lysol, bleach) after
use and after any spill of viable material. An absorbant lab mat may be used on areas in which spills are possible; place any
contaminated lab mat in the autoclave buckets.
Spills of microorganisms should be cleaned by absorption into paper towels followed by disinfection of the surface with a
suitable disinfectant. Place all soiled paper towels and gloves used during spill clean-up into autoclave buckets. If a spill is
too large for simple clean-up, such as a broken culture flask, leave a warning sign and contact the laboratory supervisor.
Contaminated clothing should be removed and replaced with emergency scrubs kept in the laboratory. Use the bag from
the scrubs to store clothing until it can be laundered (with a suitable disinfectant, such as bleach) or disposed.
If material gets on skin, wash skin thoroughly with soap and water. If material gets in the eyes, rinse for fifteen minutes
with saline solution or eye wash. Contact laboratory supervisor.

Waste disposal
Solid wastes contaminated by microorganisms or rDNA, including used gloves, pipette tips, petri dishes, and paper
products, must be placed in an autoclave bucket lined with paper towels or in an autoclavable biohazard waste bag. These
wastes should be autoclaved at the P6 setting. Autoclavable bags should be placed in a bucket or tray for autoclaving. After
autoclaving, solid wastes should be transferred to the green bin in GH10 for disposal. The bin is marked Autoclaved Solid
Wastes

Liquid cultures in containers less than 250 ml/container should be decontaminated by autoclaving at P5
or P6. Liquid cultures in containers 250 ml and up must be autoclaved at P6. Alternatively, small quantities of liquid culture
may be decontaminated by addition of one volume of bleach followed by soaking overnight. Decontaminated liquid waste
may be washed down the drain.
Sharps such as scalpel blades and glass waste must be placed in a sharps container.
Chemical wastes must be placed in appropriate waste containers. Liquid wastes should be segregated as either aqueous,
halogenic organic (like chloroform), or flammable organic waste. Solid wastes should be segregated as hazardous or non-
hazardous. Gels containing ethidium bromide or similar mutagens should be placed in a labeled plastic bag and allowed to
dry. Buffers containing ethidium bromide or similar mutagens should be passed through a sealed charcoal filter. Used filters
and dried gels can be submitted to the Environmental Programs

Recombinant Protein Production

Once a gene has been cloned, the protein it encodes can be produced in large amounts with relative
ease.

Successful expression of cloned genes depends on several factors.

Obviously, bacterial genes will usually be expressed when carried on cloning vectors in bacterial host
cells, provided that they are next to a suitable bacterial promoter.

Special plasmids known as expression vectors are often used to enhance gene expression. providing a
strong promoter drives expression of the cloned gene

Expression vectors also contain genes for antibiotic resistance to allow selection of the vector and
therefore the recombinant protein. In addition, they must have an origin of replication appropriate to
the host.

The expression of eukaryotic proteins is more problematic. Although eukaryotic cells can be used to
express eukaryotic proteins, bacteria are simpler to grow and manipulate genetically. Therefore it is
often desirable to express eukaryotic
proteins in bacteria

Because eukaryotic promoters do not work in bacterial cells, it is necessary to provide a bacterial
promoter.

In addition, bacteria cannot process introns; therefore it is standard procedure to clone the cDNA
version of eukaryotic genes, which lacks the introns and consists solely of uninterrupted coding
sequence.

In fact, the cDNA version of eukaryotic genes is generally used, even for expression in eukaryotic
cells, not only to avoid possible processing problems, but also because the amount of DNA is much
smaller and consequently easier to handle.
AVOIDING TOXIC EFFECTS OF PROTEIN OVERPRODUCTION
Although higher yields are usually desirable, overproduction of a recombinant protein may harm the
host cell. In bacteria, when too much protein is manufactured too fast, the surplus forms inclusion
bodies. These are dense crystals of misfolded and nonfunctional protein.

Thus expression systems for recombinant proteins have features to control when and how much
protein the host cell makes.

Two common expression systems are the pET and pBAD systems for E. coli. These have control
mechanisms to switch recombinant protein production
on or off, and the pBAD system can also modulate the amount of protein produced.

IMPROVING PROTEIN SECRETION

When a bacterial cell synthesizes a recombinant protein, it may end up in the cytoplasm, the
periplasmic space between the inner and outer membranes, or be exported out of the cell into the
culture medium.

Secretion across the inner, cytoplasmic, membrane is directed by the presence of a hydrophobic signal
sequence at the N-terminal end of the newly synthesized protein.

The signal sequence is cut off after export by signal peptidase (also known as leader peptidase).
Although bacteria such as E. coli export few proteins, special
secretion systems do exist that allow export of proteins across both membranes into the culture
medium.

PROTEIN FUSION EXPRESSION VECTORS

Joining the coding sequences of two proteins together in frame makes a protein fusion Protein fusions
also help address the issues of stability and purification.

Many eukaryotic proteins are unstable inside the bacterial cell. This is especially true of growth
factors, hormones, and regulatory peptides, which are often too short to fold into stable 3D
configurations.

Attaching them to the C terminus of a stable bacterial protein protects them from degradation. If the
carrier protein is carefully chosen, purification may be greatly facilitated.

EXPRESSION OF PROTEINS BY EUKARYOTIC CELLS

Although bacterial cells have successfully expressed many eukaryotic proteins, there are cases where
it is best to express eukaryotic proteins using eukaryotic cells.

Some eukaryotic proteins are unstable or inactive after being made by bacterial cells. This is
especially true of proteins that require posttranslational modification.
A variety of eukaryotic modifications may occur after the polypeptide chain has been made These
include:
(a) Chemical modifications that form novel amino acids in the polypeptide chain.

(b) Formation of disulfide bonds between correct cysteine partners (e.g., the assembly of insulin,

(c) Glycosylation, that is, the addition of sugar residues at specific locations on the protein. Many cell
surface proteins are glycosylated and will not assemble correctly into membranes or function properly
if lacking their glycosyl components.
(d) Addition of a variety of extra groups, such as fatty acid chains, acetyl groups, phosphate groups,
sulfate groups.

(e) Cleavage of precursor proteins. This may occur in several stages, as illustrated by insulin.
Cleavage may be involved with secretion, correct folding,
and/or activation of proteins.

The enzymes required for modification and processing are normally absent from bacterial cells,
making it necessary to express eukaryotic proteins in eukaryotic cells.

Related processing enzymes are often present in a range of higher organisms; thus it is rarely
absolutely necessary to express a protein in its original organism.

It is now possible to engineer whole transgenic animals or plants to produce recombinant proteins. A
further advantage of expressing eukaryotic proteins in eukaryotic cells is that contamination with
bacterial components is avoided.

Proteins Produced by Recombinant Technology

Products from transgenic animals

Transgenic animals such as cattle, sheep, goats, pigs and even rabbits have several significant
advantages for the production of recombinant proteins over other systems, including their potential for
large-scale production, correct glycosylation patterns and post-translational modifications, low
running costs, rapid propagation of the transgenic founders and high expression stability.

The most promising site for production of recombinant proteins is the mammary gland, but other body
fluids including blood, urine and seminal fluid have also been explored

Therapeutic antibodies

Antibodies for human therapy are the fastest growing set of new biopharmaceuticals, in terms of
number of products and market share.

20 antibody products are approved for therapeutic use and several hundred are undergoing trials for
applications in cancer therapy, autoimmune diseases, transplantation, antibiotic resistant infections,
biodefense and immune deficiencies

The first therapeutic monoclonal to gain regulatory approval was Ortho Biotechs orthoclone OKT3
(Muromonab) antibody to T-cell CD3 antigen for the suppression of kidney transplant rejection in
June 1986

Polyclonals are also more effective than monoclonals in the formation of immune complexes
responsible for complement activation and the recruitment of neutrophils and macrophages

Kirin Pharma have created transchromosomic cattle using a human artificial chromosome vector
containing the entire un-rearranged sequences of the human immunoglobulin G heavy-chain and
lambda light-chain loci

Blood clotting factor

Molly and Polly both carried the human gene that codes for a protein called Factor IX. Factor IX is a
blood clotting protein, and it is commonly used to treat patients that have hemophilia B, a disease that
is caused by a deficiency in Factor IX. The goal of creating Molly and Polly was to produce a herd of
sheep that produced Factor IX in their milk. The only other source of Factor IX is from human blood
plasma. By producing Factor IX in sheep milk, it is hoped that the cost of production will be greatly
reduced.

Serum albumin

Advanced Cell Technology, Inc is using the queen of milk production, the cow, for potential use as
bioreactors. They have produced transgenic cows that secrete the protein serum albumin in their milk,
a protein that is used to extend blood volume and is used in patients suffering from traumatic injuries,
such as burns

PHARMACOLOGICAL PRODUCTSBY BACTERIAL GENETIC ENGINEERING

Recombinant DNA techniques have modified the genetic nature of microorganisms and are used to
produce various compounds that are important for Industries, Agriculture, Environmental
management and pharmaceuticals.

Recombinant DNA microorganisms can produce compounds 1000 fold higher than isolated from
plants and animals, or synthesized by chemists.
Antibiotics Calvulanic acid, Daptomycon, Fosfomycin, Novobiocin, Tylectol and Cubebol were
produced from genetically engineered Streptomyces. The engineered E.Coli produced
Deoxyerythromycin, Magnoflorine and Torulene

Human alpha hemoglobin , Gonodotrophine releasing hormone, human growth hormone, human
parathyroid hormone and human interferon were produced from E.Coli cells. Production of human
insulin was achieved in E.Coli.

Recombinant products from Plants

The exploitation of plant biotechnology to produce diagnostic and therapeutic products has become a
well-recognized and important field of biopharmaceutical science with promising economic potential.

whole plants and plant cell cultures are now considered as viable and competitive systems for large-
scale production of industrial, pharmaceutical recombinant proteins and secondary metabolites.

Recombinant protein production using transgenic plants as bioreactors is likely to be more economical
than alternative systems, especially for large-scale needs.

Although tobacco leaf has been the most popular expression system for recombinant antibodies,
several groups have explored the use of other plants and plant seeds for use in expression and large-
scale production

corn seeds have shown particular utility in the production of high levels of secretory immunoglobulin
A (sIgA), the most abundant antibody class produced by the body

The plant secretory antibody (Tobaco plant) afforded specific protection in humans against oral
streptococcal colonization for at least 4 month

Avicidin is a full-length antibody specific for EpCAM (a marker of colorectal cancer) developed
jointly by NeoRx and Monsanto

The idea for transgenic plant-derived vaccines originated in the early 1990s. At the time, Charles
Arntzen and his colleagues envisaged a cost-effective vaccine production system with a safe and
efficacious delivery system through the use of plants specifically engineered to deliver safe subunit
preparations of candidate antigens for major diseases afflicting developing and developed nations

The first demonstration of vaccine antigen expression was reported in 1990 when R.I. Curtiss and
C.A. Cardineau expressed the Streptococcus mutans surface protein antigen A (SpaA) in tobacco.
After feeding the transgenic tobacco tissue to mice, the researchers observed that a mucosal immune
response was induced in response to the SpaA protein.

Subsequently C. Arntzen and his collaborators provided proof of concept for what is now referred to
as edible vaccines by showing that the surface antigen of Hepatitis B could be synthesized in
transgenic plants

Since these initial reports, numerous plant species have been used for antigen expression, including
tobacco, potato, tomato, banana, corn, lupine, and lettuce

Secondary metabolites of pharmacological value

The plasticity of plant metabolic activity is most evident in the wide variety of secondary metabolites
accumulated by plants in their leaves, roots, and other organs
Within recent times, many plantderived products for treating human disorders have reached the
market place as useful drugs including atropine, hyoscyamine, scopolamine, taxol (anticancer),
vinblastine/vincristine, artemisinin (antimalarial), reserpine (antihypertension), and quinine
(antimalaria).

The ability to genetically engineer plant genomes has allowed for the direct manipulation of plant
metabolism and the potential for manipulating the content and nature of plant secondary metabolites
of commercial value. Plants are now being considered as potential factories for the production of a
variety of useful compounds

Recombinant Protein Production

Once a gene has been cloned, the protein it encodes can be produced in large
amounts with relative ease.

Successful expression of cloned genes depends on several factors.

Obviously, bacterial genes will usually be expressed when carried on cloning

vectors in bacterial host cells, provided that they are next to a suitable bacterial
promoter.

Special plasmids known as expression vectors are often used to enhance gene
expression. providing a strong promoter drives expression of the cloned gene

Expression vectors also contain genes for antibiotic resistance to allow selection
of the vector and therefore the recombinant protein. In addition, they must have
an origin of replication appropriate to the host.

The expression of eukaryotic proteins is more problematic. Although eukaryotic

cells can be used to express eukaryotic proteins, bacteria are simpler to grow
and manipulate genetically. Therefore it is often desirable to express eukaryotic
proteins in bacteria

Because eukaryotic promoters do not work in bacterial cells, it is necessary to

provide a bacterial promoter.

In addition, bacteria cannot process introns; therefore it is standard procedure to

clone the cDNA version of eukaryotic genes, which lacks the introns and
consists solely of uninterrupted coding sequence.

In fact, the cDNA version of eukaryotic genes is generally used, even for
expression in eukaryotic cells, not only to avoid possible processing problems,
but also because the amount of DNA is much smaller and consequently easier to
handle.
AVOIDING TOXIC EFFECTS OF PROTEIN OVERPRODUCTION
Although higher yields are usually desirable, overproduction of a recombinant
protein may harm the host cell. In bacteria, when too much protein is
manufactured too fast, the surplus forms inclusion bodies. These are dense
crystals of misfolded and nonfunctional protein.

Thus expression systems for recombinant proteins have features to control when
and how much protein the host cell makes.

Two common expression systems are the pET and pBAD systems for E. coli.
These have control mechanisms to switch recombinant protein production
on or off, and the pBAD system can also modulate the amount of protein
produced.

IMPROVING PROTEIN SECRETION

When a bacterial cell synthesizes a recombinant protein, it may end up in the
cytoplasm, the periplasmic space between the inner and outer membranes, or be
exported out of the cell into the culture medium.

Secretion across the inner, cytoplasmic, membrane is directed by the presence

of a hydrophobic signal sequence at the N-terminal end of the newly
synthesized protein.

The signal sequence is cut off after export by signal peptidase (also known as
leader peptidase). Although bacteria such as E. coli export few proteins, special
secretion systems do exist that allow export of proteins across both membranes
into the culture medium.

PROTEIN FUSION EXPRESSION VECTORS

Joining the coding sequences of two proteins together in frame makes a protein
fusion Protein fusions also help address the issues of stability and purification.

Many eukaryotic proteins are unstable inside the bacterial cell. This is
especially true of growth factors, hormones, and regulatory peptides, which are
often too short to fold into stable 3D configurations.

Attaching them to the C terminus of a stable bacterial protein protects them

from degradation. If the carrier protein is carefully chosen, purification may be
greatly facilitated.

EXPRESSION OF PROTEINS BY EUKARYOTIC CELLS

Although bacterial cells have successfully expressed many eukaryotic proteins,
there are cases where it is best to express eukaryotic proteins using eukaryotic
cells.

Some eukaryotic proteins are unstable or inactive after being made by bacterial
cells. This is especially true of proteins that require posttranslational
modification.
A variety of eukaryotic modifications may occur after the polypeptide chain has
been made These include:
(a) Chemical modifications that form novel amino acids in the polypeptide
chain.

(b) Formation of disulfide bonds between correct cysteine partners (e.g., the
assembly of insulin,

(c) Glycosylation, that is, the addition of sugar residues at specific locations on
the protein. Many cell surface proteins are glycosylated and will not assemble
correctly into membranes or function properly if lacking their glycosyl
components.

(d) Addition of a variety of extra groups, such as fatty acid chains, acetyl
groups, phosphate groups, sulfate groups.

(e) Cleavage of precursor proteins. This may occur in several stages, as

illustrated by insulin. Cleavage may be involved with secretion, correct folding,
and/or activation of proteins.

The enzymes required for modification and processing are normally absent from
bacterial cells, making it necessary to express eukaryotic proteins in eukaryotic
cells.

Related processing enzymes are often present in a range of higher organisms;

thus it is rarely absolutely necessary to express a protein in its original
organism.

It is now possible to engineer whole transgenic animals or plants to produce

recombinant proteins. A further advantage of expressing eukaryotic proteins in
eukaryotic cells is that contamination with bacterial components is avoided.
Proteins Produced by Recombinant Technology

Products from transgenic animals

Transgenic animals such as cattle, sheep, goats, pigs and even rabbits have
several significant advantages for the production of recombinant proteins over
other systems, including their potential for large-scale production, correct
glycosylation patterns and post-translational modifications, low running costs,
rapid propagation of the transgenic founders and high expression stability.

The most promising site for production of recombinant proteins is the mammary
gland, but other body fluids including blood, urine and seminal fluid have also
been explored

Therapeutic antibodies

Antibodies for human therapy are the fastest growing set of new
biopharmaceuticals, in terms of number of products and market share.

20 antibody products are approved for therapeutic use and several hundred are
undergoing trials for applications in cancer therapy, autoimmune diseases,
transplantation, antibiotic resistant infections, biodefense and immune
deficiencies
The first therapeutic monoclonal to gain regulatory approval was Ortho
Biotechs orthoclone OKT3 (Muromonab) antibody to T-cell CD3 antigen for
the suppression of kidney transplant rejection in June 1986

Polyclonals are also more effective than monoclonals in the formation of

immune complexes responsible for complement activation and the recruitment
of neutrophils and macrophages

Kirin Pharma have created transchromosomic cattle using a human artificial

chromosome vector containing the entire un-rearranged sequences of the human
immunoglobulin G heavy-chain and lambda light-chain loci

Blood clotting factor

Molly and Polly both carried the human gene that codes for a protein called
Factor IX. Factor IX is a blood clotting protein, and it is commonly used to treat
patients that have hemophilia B, a disease that is caused by a deficiency in
Factor IX. The goal of creating Molly and Polly was to produce a herd of sheep
that produced Factor IX in their milk. The only other source of Factor IX is
from human blood plasma. By producing Factor IX in sheep milk, it is hoped
that the cost of production will be greatly reduced.

Serum albumin

Advanced Cell Technology, Inc is using the queen of milk production, the cow,
for potential use as bioreactors. They have produced transgenic cows that
secrete the protein serum albumin in their milk, a protein that is used to extend
blood volume and is used in patients suffering from traumatic injuries, such as
burns

PHARMACOLOGICAL PRODUCTSBY BACTERIAL GENETIC

ENGINEERING

Recombinant DNA techniques have modified the genetic nature of

microorganisms and are used to produce various compounds that are important
for Industries, Agriculture, Environmental management and pharmaceuticals.

Recombinant DNA microorganisms can produce compounds 1000 fold higher

than isolated from plants and animals, or synthesized by chemists.
Antibiotics Calvulanic acid, Daptomycon, Fosfomycin, Novobiocin, Tylectol
and Cubebol were produced from genetically engineered Streptomyces. The
engineered E.Coli produced Deoxyerythromycin, Magnoflorine and Torulene

Human alpha hemoglobin , Gonodotrophine releasing hormone, human growth

hormone, human parathyroid hormone and human interferon were produced
from E.Coli cells. Production of human insulin was achieved in E.Coli.

Recombinant products from Plants

The exploitation of plant biotechnology to produce diagnostic and therapeutic

products has become a well-recognized and important field of
biopharmaceutical science with promising economic potential.

whole plants and plant cell cultures are now considered as viable and
competitive systems for large-scale production of industrial, pharmaceutical
recombinant proteins and secondary metabolites.

Recombinant protein production using transgenic plants as bioreactors is likely

to be more economical than alternative systems, especially for large-scale
needs.

Although tobacco leaf has been the most popular expression system for
recombinant antibodies, several groups have explored the use of other plants
and plant seeds for use in expression and large-scale production

corn seeds have shown particular utility in the production of high levels of
secretory immunoglobulin A (sIgA), the most abundant antibody class produced
by the body

The plant secretory antibody (Tobaco plant) afforded specific protection in

humans against oral streptococcal colonization for at least 4 month

Avicidin is a full-length antibody specific for EpCAM (a marker of colorectal

cancer) developed jointly by NeoRx and Monsanto

The idea for transgenic plant-derived vaccines originated in the early 1990s. At
the time, Charles Arntzen and his colleagues envisaged a cost-effective vaccine
production system with a safe and efficacious delivery system through the use
of plants specifically engineered to deliver safe subunit preparations of
candidate antigens for major diseases afflicting developing and developed
nations
The first demonstration of vaccine antigen expression was reported in 1990
when R.I. Curtiss and C.A. Cardineau expressed the Streptococcus mutans
surface protein antigen A (SpaA) in tobacco. After feeding the transgenic
tobacco tissue to mice, the researchers observed that a mucosal immune
response was induced in response to the SpaA protein.

Subsequently C. Arntzen and his collaborators provided proof of concept for

what is now referred to as edible vaccines by showing that the surface antigen
of Hepatitis B could be synthesized in transgenic plants

Since these initial reports, numerous plant species have been used for antigen
expression, including tobacco, potato, tomato, banana, corn, lupine, and lettuce

Secondary metabolites of pharmacological value

The plasticity of plant metabolic activity is most evident in the wide variety of
secondary metabolites accumulated by plants in their leaves, roots, and other
organs

Within recent times, many plantderived products for treating human disorders
have reached the market place as useful drugs including atropine, hyoscyamine,
scopolamine, taxol (anticancer), vinblastine/vincristine, artemisinin
(antimalarial), reserpine (antihypertension), and quinine (antimalaria).

The ability to genetically engineer plant genomes has allowed for the direct
manipulation of plant metabolism and the potential for manipulating the content
and nature of plant secondary metabolites of commercial value. Plants are now
being considered as potential factories for the production of a variety of useful
compounds

Safety Guideline for Recombinant DNA and Disposal

The new capabilities to manipulate the genetic material present tremendous
potential and find use in many novel experiments and applications. These
developments have generated a sense of concern among scientists working in
biological areas and others to find ways how safely the research in the field
should be carried out and means to regulate work involving pathogenic
microorganisms and genes of virulence.

A large number of research institutions in Government, Universities and private

R&D labs have active biotech programmes where research is being done in both
in basic and applied fronts utilising microorganisms plant and animals, tissue
culture and cell lines and on development of vaccines towards communicable
diseases of both men and animals. A good deal of effort is being made in the
areas of diagnostics, biofertilizers, biocides, fertility control, tissue culture of
high value crops to develop technologies and useful products.

The guidelines cover areas of research involving genetically engineered

organism. It also deals with genetic transformation of green plants, rDNA
technology in vaccine development and on large scale production and
dekliberate/ accidental release of organisms, plants, animals and products
derived by rDNA technology into the environment.

The guidelines are in respect of safety measures for the research activities, large
scale use and also the environmental impact during field applications of
genetically altered material products

Research: The levels of the risk and the classification of the organisms within
these levels based on pathogenicity and local prevalence of diseases and on
epidemic causing strains Some of the microorganisms not native to the country
have been assigned to a special category requiring highest degree of safety.
The guidelines employ the concept of physical and biological containment and
also based upon the principle of good laboratory practice (GLP).

Large scale operations: The concern does not diminish when it comes to the
use of recombinant organisms scale fermentation operations on large scale
fermentation operations or applications of it in the environment. As such, the
guidelines prescribe criteria for good large scale practices (GLSP) for using
recombinant organisms. These include measures such as proper engineering for
containment, quality control, personnel protection, medical surveillance, etc.

Environmental risks: Application and release of engineered organisms into the

environment could lead to ecological consequences and potential risks unless
necessary safeguards are taken into account. The guidelines prescribe the
criteria for assessment of the ecological aspects on a case by case basis for
planned introduction of rDNA organism into the environment. It also suggests
regulatory measures to ensure safety for import of genetically engineered
materials, plants and animals. The recommendations also cover the various
quality control methods needed to establish the safety, purity and efficacy of
rDNA products.

GUIDELINES

Classification of a pathogenic microorganisms

 The classification of infective microorganisms are drawn up under 4 risk
groups in increasing order of risk
Based on the risk assessment information, the probability of risk could be
further assigned certain quantitative values
Containment
Containment facilities for different Risk Groups as per the recommendations of
World Health Organization (WHO)
The term "Containment" is used in describing the safe methods for managing
infectious agents in the laboratory environment where they are being handled or
maintained.

Purpose of containment
To reduce exposure of laboratory workers, other persons, and outside
environment to potentially hazardous agents.
Types of containment
Biological containment (BC):

In consideration of biological containment, the vector (plasmid, organelle, or

virus) for the recombinant DNA and the host (bacterial, plant, or animal cell) in
which the vector is propagated in the laboratory will be considered together.

Any combination of vector and host which is to provide biological containment

must be chosen or constructed to limit the infectivity of vector to specific hosts
and control the host-vector survival in the environment.

These have been categorized into two levels - one permitting standard
biological containment and the other even higher that relates to normal and
disabled host-vector systems respectively

Physical Containment (PC):

The objective of physical containment is to confine recombinant organisms
thereby preventing the exposure of the researcher and the environment to the
harmful agents. Physical containment is achieved through the use of
i) Laboratory Practice,
ii) Containment
Equipment, and
iii) Special Laboratory Design.

The protection of personnel and the immediate laboratory environment from

exposure to infectious agents, is provided by good microbiological techniques
and the use of appropriate safety equipment, (Primary Containment).
The protection of the environment external to the laboratory from exposure to
infectious materials, is provided by a combination of facility design and
operational practices, (Secondary Containment).

Elements of Containment:

The three elements of containment include laboratory practice and

technique, safety equipment and facility design.
i) Laboratory practice and technique:
Strict adherence to standard microbiological practices and techniques
Awareness of potential hazards
Providing/arranging for appropriate training of personnel
Selection of safety practices in addition to standard laboratory practices if
required
Developing of adopting a biosafety or operations manual which identifies the
hazards

ii) Safety equipment (primary barriers): Safety equipment includes biological

safety cabinets and a variety of enclosed containers (e.g. safety centrifuge cup).
The biological safety cabinet (BSC) is the principal device used to provide
containment of infectious aerosols generated by many microbiological
procedures. Three types of BSCs (Class I, II, III) are used in microbiological
laboratories. Safety equipment also includes items for personal protection
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields and
safety glasses,
etc.
iii) Facility Design (Secondary barriers): The design of the facility is important
in providing a barrier to protect persons working in the facility but outside of
the laboratory and those in the community from infectious agents which may be
accidentally released from the laboratory.
There are three types of facility designs: viz, the Basic Laboratory (for Risk
Group I and II), the Containment Laboratory (for Risk Group III) and the
Maximum Containment Laboratory
(for Risk Group IV).

MECHANISM OF IMPLEMENTATION OF BIOSAFETY GUIDELINES

For implementation of the guidelines it is necessary to have an institutional mechanism to
ensure the compliance of requisite safeguards at various levels. The guidelines prescribe
specific actions that include establishing safety procedures for rDNA research, production
and release to the environment and setting up containment conditions for certain experiments.
The guidelines suggest compliance of the safeguards through voluntary as well as regulatory
approach. In this connection, it is proposed to have a mechanism of advisory and regulatory
bodies to deal with the specific and discretionary actions on the following:
a. Self regulation and control in the form of guidelines on recombinant research activities
b. Regulation of large scale use of engineered organisms in production activity and release of
organisms in
environmental applications under statutory provisions. The institutional mechanism as
proposed for implementation of guidelines is shown in organogram in Figure 2.
Mainly it consists of the following:-
i) Recombinant DNA Advisory Committee (RDAC)
ii) Institutional Biosafety Committee (IBSC)
iii) Review Committee on Genetic Manipulation (RCGM)
iv) Genetic Engineering Approval Committee (GEAC)

Recombinant DNA Advisory Committee (RDAC):

The Committee should take note of developments at national and international levels in
Biotechnology towards the currentness of the safety regulation for India on recombinant
research use and applications. It would meet once in 6
months or sooner for this purpose.
The specific terms of reference for Recombinant Advisory Committee include the following :
i) To evolve long term policy for research and development in Recombinant DNA research.
ii) To formulate the safety guidelines for Recombinant DNA Research to be followed in
India.
iii) To recommended type of training programme for technicians and research fellows for
making
them adequately aware of hazards and risks involved in recombinant DNA research and
methods of avoiding it.
Institutional Biosafety Committee (IBSC)
Institutional Biosafety Committee (IBSC) are to be constituted in all centres
engaged in genetic engineering research and production activities. The
Committee will constitute the following:
(i) Head of the Institution or nominee
(ii) 3 or more scientists engaged in DNA work or molecular biology with an
outside expert in the relevant discipline.
(iii) A member with medical qualifications - Biosafety Officer (in case of work
with pathogenic agents/large scale use).
(iv) One member nominated by DBT.
The Institutional Biosafety Committee shall be the nodal point for interaction
within institution for implementation of the guidelines. Any research project
which is likely to have biohazard potential (as envisaged by the guidelines)
during the execution stage or which involve the production of either
microorganisms or biologically active molecules that might cause bio-hazard
should be notified to IBSC. IBSC will allow genetic engineering activity on
classified organisms only at places where such work should be performed as per
guidelines. Provision of suitable safe storage facility of donor, vectors,
recipients and other materials involved in experimental work should be made
and may be subjected to inspection on accountability.

Review Committee on Genetic Manipulation (RCGM):

 The RCGM will have the following composition:
i) Department of Biotechnology
ii) Indian Council of Medical Research
iii) Indian Council of Agricultural Research
iv) Council of Scientific & Industrial Research
v) Three Experts in Individual capacity
vi) Department of Science & Technology
The RCGM will have the functions:
i) To establish procedural guidance manual - procedure for regulatory process
with respect to activity involving genetically engineered organisms in research,
production and applications related to environmental safety.
ii) To review the reports in all approved ongoing research projects involving
high risk category and controlled field experiments, to ensure that safeguards
are maintained as per guidelines.
iii) To recommended the type of containment facility and the special
containment conditions to be followed for experimental trials and for certain
experiments.
iv) To advise customs authorities on import of biologically active material,
genetically engineered substances or products and on excisable items to Central
Revenue and Excise.
v) To assist Department of Industrial Development, Banks towards clearance of
applications in setting up industries based on genetically engineered organisms.
vi) To assist the Bureau of Indian Standards to evolve standards for biologics
produced by rDNA technology.
vii) To advise on intellectual property rights with respect to rDNA technology
on patents

Genetic Engineering Approval Committee (GEAC):

Genetic Engineering Approval Committee (GEAC) will function under the
Department of Environment (DOEn) as statutory body for review and approval
of activities involving large scale use of genetically engineered organisms and
their products in research and development, industrial production,
environmental release and field applications.
The functions include giving approval from environmental angle on:
i) Import, export, transport, manufacture, process, selling of any
microorganisms or genetically engineered substances or cells including food
stuffs and additives that contains products derived by Gene Therapy.
ii) Discharge of Genetically engineered/classified organisms/cells from
Laboratory, hospitals and related areas into environment.
iii) Large scale use of genetically engineered organisms/classified
microorganisms in industrial production and applications. (Production shall not
be commenced without approval).
iv) Deliberate release of genetically engineered organisms. The approval will be
for a period of 4 years.

Institutional mechanism for implementation of guidelines frame work for

implementation

GOI - Government of India, DBT - Department of Biotechnology

RDAC - Recombinant DNA Advisory Committee, IBSC - Institutional Biosafety Committee
RCGM - Review Committee on Genetic Manipulation, DOEn - Department of Environment
GEAC - Genetic Engineering Approval Committee SBCC - State Biotechnology Coordination
Committee, PI - Principal Invstigator (R&D/Industry/Others)
FA - Funding Agency (Govt./Private & Public Institutions

Safety Precautions for Laboratories Using recombinant DNA (rDNA)

Laboratory Access
Only authorized persons may enter the laboratory. An authorized person must
understand the safety precautions of a recombinant DNA laboratory or be
accompanied by one who does. Laboratory doors should be kept closed and
locked except when a laboratory worker is present. All entrances to the
laboratory from the hallway should be marked with contact information
regarding the faculty member(s) in charge for the use of emergency personnel
Protective clothing
Goggles or safety glasses should be worn in the laboratory by all personnel at
all times.
Closed Toe Footware should be worn in the laboratory by all personnel at all
times. No sandals, flip-flops, etc.
Laboratory coats should be worn in the laboratory whenever rDNA work is
being done. Do not wear these coats into areas in which food or drink may be
consumed. Isolate them in a plastic bag when taking them to the laundry.
Disposable gloves must be worn when handling microorganisms or nucleic
acids. Change gloves immediately after obvious contamination or tears. Do not
leave the laboratory and handle door-knobs, etc. while still wearing gloves.
Wash your hands before leaving.
Goggles or safety glasses with a UV-protective coating are required whenever
gels are observed on an ultraviolet transilluminator. Both goggles AND a face
shield are required for longer exposures, such as cutting out bands. Eye damage
and serious facial sunburn may result if these precautions are ignored.
Masks should be worn when weighing out or cleaning up dangerous powders
such as ethidium bromide, acrylamide and SDS. Always read labels before
handling chemicals.
Laboratory behavior
Food, drink, gum-chewing, smoking, and application of cosmetics are
prohibited in the laboratory.
No food or drink may be stored in refrigerators or elsewhere in the laboratory.
Mouth pipetting is prohibited.
Work Surfaces and Spills
Bench surfaces and microcentrifuges must be disinfected with a suitable
agent (such as 70% ethanol, Lysol, bleach) after use and after any spill of viable
material. An absorbant lab mat may be used on areas in which spills are
possible; place any contaminated lab mat in the autoclave buckets.
Spills of microorganisms should be cleaned by absorption into paper towels
followed by disinfection of the surface with a suitable disinfectant. Place all
soiled paper towels and gloves used during spill clean-up into autoclave
buckets. If a spill is too large for simple clean-up, such as a broken culture flask,
leave a warning sign and contact the laboratory supervisor.
Contaminated clothing should be removed and replaced with emergency
scrubs kept in the laboratory. Use the bag from the scrubs to store clothing until
it can be laundered (with a suitable disinfectant, such as bleach) or disposed.
If material gets on skin, wash skin thoroughly with soap and water. If material
gets in the eyes, rinse for fifteen minutes with saline solution or eye wash.
Contact laboratory supervisor.

Waste disposal
Solid wastes contaminated by microorganisms or rDNA, including used gloves,
pipette tips, petri dishes, and paper products, must be placed in an autoclave
bucket lined with paper towels or in an autoclavable biohazard waste bag. These
wastes should be autoclaved at the P6 setting. Autoclavable bags should be
placed in a bucket or tray for autoclaving. After autoclaving, solid wastes
should be transferred to the green bin in GH10 for disposal. The bin is marked
Autoclaved Solid Wastes

Liquid cultures in containers less than 250 ml/container should be

decontaminated by autoclaving at P5
or P6. Liquid cultures in containers 250 ml and up must be autoclaved at P6.
Alternatively, small quantities of liquid culture may be decontaminated by
addition of one volume of bleach followed by soaking overnight.
Decontaminated liquid waste may be washed down the drain.
Sharps such as scalpel blades and glass waste must be placed in a sharps
container.
Chemical wastes must be placed in appropriate waste containers. Liquid wastes
should be segregated as either aqueous, halogenic organic (like chloroform), or
flammable organic waste. Solid wastes should be segregated as hazardous or
non-hazardous. Gels containing ethidium bromide or similar mutagens should
be placed in a labeled plastic bag and allowed to dry. Buffers containing
ethidium bromide or similar mutagens should be passed through a sealed
charcoal filter. Used filters and dried gels can be submitted to the
Environmental Programs