Vectors for Gene Transformation

Sathiyaraj Srinivasan
 2006315223

Viral vector

Viral vectors are a tool commonly used by molecular biologists to deliver

genetic material into cells. This process can be performed inside a living
organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized
molecular mechanisms to efficiently transport their genomes inside the cells they
infect. Delivery of genes by a virus is termed transduction and the infected cells
are described as transduced. Molecular biologists first harnessed this machinery
in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the
bacteriophage lambda to infect monkey kidney cells maintained in culture.

Key properties of viral vector 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.
• 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.

Applications
Basic research

Viral vectors were originally developed as an alternative to transfection of naked

DNA for molecular genetic experiments. Compared to traditional methods such
as calcium phosphate precipitation, transduction can ensure that nearly 100% of
cells are infected without severely affecting cell viability. Furthermore, some
viruses integrate into the cell genome facilitating stable expression. However,
transfection is still the method of choice for many applications as construction of
a viral vector is a much more laborious process. Protein coding genes can be
expressed using viral vectors, commonly to study the function of the particular
protein. Viral vectors, especially retroviruses, stably expressing marker genes
such as GFP are widely used to permanently label cells to track them and their
progeny, for example in xenotransplantation experiments, when cells infected in
vitro are implanted into a host animal. Genes inserted into the vector can encode
shRNAs and siRNAs used to efficiently block or silence production of a specific
protein. Such knock-down experiments are much quicker and cheaper to carry out
than gene knockout. But as the silencing is sometimes non-specific and has off-
target effects on other genes, it provides less reliable results.

Gene therapy

In the future gene therapy may provide a way to cure genetic disorders, such as
severe combined immunodeficiency or cystic fibrosis. Several gene therapy trials
have used viruses to deliver 'good' genes to the cells of the patient's body. There
have been a huge number of laboratory successes with gene therapy. However,
several problems of viral gene therapy must be overcome before it gains
widespread use. Immune response to viruses not only impedes the delivery of
genes to target cells but can cause severe complications for the patient. In one of
the early gene therapy trials in 1999 this led to the death of Jesse Gelsinger, who
was treated using an adenoviral vector.

Some viral vectors, for instance lentiviruses, insert their genomes at a seemingly
random location on one of the host chromosomes, which can disturb the function
of cellular genes and lead to cancer. In a severe combined immunodeficiency
retroviral gene therapy trial conducted in 2002, two of the patients developed
leukemia as a consequence of the treatment.[3] Adeno-associated virus-based
vectors are much safer in this respect as they always integrate at the same site in
the human genome.

Vaccines

Viruses expressing pathogen proteins are currently being developed as vaccines

against these pathogens, based on the same rationale as DNA vaccines. T-
lymphocytes recognize cells infected with intracellular parasites based on the
foreign proteins produced within the cell. T cell immunity is crucial for
protection against viral infections and such diseases as malaria. A viral vaccine
induces expression of pathogen proteins within host cells similarly to the Sabin
Polio vaccine and other attenuated vaccines. However, since viral vaccines
contain only a small fraction of pathogen genes, they are much safer and sporadic
infection by the pathogen is impossible. Adenoviruses are being actively
developed as vaccines.

Types of viral vectors

Retroviruses

Retroviruses are the one of mainstays of current gene therapy approaches. The
recombinant retroviruses such as the Moloney murine leukemia virus have the
ability to integrate into the host genome in a stable fashion. They contain a
reverse transcriptase which allows integration into the host genome. They have
been used in a number of FDA-approved clinical trials such as the SCID-X1
trial.[4] The primary drawback to use of retroviruses such as the Moloney
retrovirus involves the requirement for cells to be actively dividing for
transduction. As a result, cells such as neurons are very resistant to infection and
transduction by retroviruses. There is a concern for insertional mutagensis due to
the integration into the host genome which can lead to cancer or leukemia.

Lentiviruses
Packaging and infection by a lentiviral vector

Lentiviruses are a subclass of Retroviruses. They are widely adapted as vectors

thanks to their ability integrate into the genome of non-dividing as well as
dividing cells. The viral genome in the form of RNA is reverse-transcribed when
the virus enters the cell to produce DNA, which is then inserted into the genome
at a random position by the viral integrase enzyme. The vector, now called a
provirus, remains in the genome and is passed on to the progeny of the cell when
it divides. The site of integration is unpredictable, which can pose a problem. The
provirus can disturb the function of cellular genes and lead to activation of
oncogenes promoting the development of cancer, which raises concerns for
possible applications of lentiviruses in gene therapy.

For safety reasons lentiviral vectors never carry the genes required for their
replication. To produce a lentivirus, several plasmids are transfected into a so-
called packaging cell line, commonly HEK 293. One or more plasmids,
generally referred to as packaging plasmids, encode the virion proteins, such as
the capsid and the reverse transcriptase. Another plasmid contains the genetic
material to be delivered by the vector. It is transcribed to produce the single-
stranded RNA viral genome and is marked by the presence of the ψ (psi)
sequence. This sequence is used to package the genome into the virion.

Adenoviruses

As opposed to lentiviruses, adenoviral DNA does not integrate into the genome
and is not replicated during cell division. This limits their use in basic research,
although adenoviral vectors are occasionally used in in vitro experiments. Their
primary applications are in gene therapy and vaccination. Since humans
commonly come in contact with adenoviruses, which cause respiratory,
gastrointestinal and eye infections, they trigger a rapid immune response with
potentially dangerous consequences. To overcome this problem scientists are
currently investigating adenoviruses to which humans do not have immunity.

Nanoengineered Substances

Substances such as Ormosil have been successfully used as a DNA vector.

Plasmid

A 'plasmid' is a DNA molecule separate from the chromosomal DNA and

capable of autonomous replication. In many cases, It is typically circular and
double-stranded. It usually occurs in bacteria, and is sometimes found in
eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

The size of plasmids varies from 1 to over 400 kilobase pairs (kbp). There may
be one copy, for large plasmids, to hundreds of copies of the same plasmid in a
single cell, or even thousands of copies, for certain artificial plasmids selected for
high copy number (such as the pUC series of plasmids). Plasmids can be part of
the mobilome, since they are often associated with conjugation, a mechanism of
horizontal gene transfer.

The term plasmid was first introduced by the American molecular biologist
Joshua Lederberg in 1952.

Antibiotic resistance

Figure 2: Illustration of a plasmid with antibiotic

resistances.
1 & 2 Genes that code for resistance. 3 Ori.
Plasmids often contain genes or gene cassettes that confer a selective advantage
to the bacterium harboring them, such as the ability to make the bacterium
antibiotic resistant.

Every plasmid contains at least one DNA sequence that serves as an origin of
replication, or ori (a starting point for DNA replication), which enables the
plasmid DNA to be duplicated independently from the chromosomal DNA
(Figure 2). The plasmids of most bacteria are circular, like the plasmid depicted
in Figure 2, but linear plasmids are also known, which superficially resemble the
chromosomes of most eukaryotes.

Episomes

An episome is a plasmid that can be added, without integration, to the

chromosomal DNA of the host organism (Fig. 3). In this situation, it can stay
intact for a long time, be either diluted out or be duplicated with every cell
division of the host, and become a basic part of its genetic makeup. The term is
no longer commonly used for plasmids, since it is now clear that a region of
homology with the chromosome such as a transposon will make a plasmid into
an episome. In mammalian systems, the term episome refers to a circular DNA
(such as a viral genome) that is maintained by noncovalent tethering to the host
cell chromosome. In detail, we distinguish:

• Minichromosomes (MCs) that contain all elements for replication and

segregation (telomeres, centromeres); MCs are usually obtained by
chromosome engineering (e.g. telomere-seeding, the use of site-specific
recombinases and/or the recombinase-mediated cassette exchange
concept)
• Plasmids and Minicircles that stay extra-chromosomal (episomal) in non-
or barely-dividing tissues (note: ´minicircles´ are plasmids devoid of
prokaryotic sequence parts and thereby poor in inactivating CpG tracts)
 • Replicating minicircles, i.e. plasmid derivatives that can recruit all
components necessary for autonomous replication from the host cell. They
replicate once per cell cycle and do not undergo epigenetic inactivation. In
contrast to viral circular episomes (SV40, BPV, EBV, HSV) no viral
components (proteins) are required for autonomous relpication.

Figure 3: Comparison of non-integrating plasmids (top) and episomes (bottom).

Top: Chromosomal DNA and plasmids replicated separately. Bottom:
Chromosomal DNA with integrated plasmids replicate as a single chromosome.

Vectors

Plasmids used in genetic engineering are called vectors. They are used to transfer
genes from one organism to another and typically contain a genetic marker
conferring a phenotype that can be selected for or against. Most also contain a
polylinker or multiple cloning site (MCS), which is a short region containing
several commonly used restriction sites allowing the easy insertion of DNA
fragments at this location. See Applications below.[citation needed]
Types

Figure 4: Overview of Bacterial conjugation

One way of grouping plasmids is by their ability to transfer to other bacteria.

Conjugative plasmids contain so-called tra-genes, which perform the complex
process of conjugation, the sexual transfer of plasmids to another bacterium (Fig.
4). Non-conjugative plasmids are incapable of initiating conjugation, hence they
can only be transferred with the assistance of conjugative plasmids, by 'accident'.
An intermediate class of plasmids are mobilizable, and carry only a subset of the
genes required for transfer. They can 'parasitise' a conjugative plasmid,
transferring at high frequency only in its presence. Plasmids are now being used
to manipulate DNA and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell. Seven

different plasmids have been found in E. coli. But related plasmids are often
incompatible, in the sense that only one of them survives in the cell line, due to
the regulation of vital plasmid functions. Therefore, plasmids can be assigned
into compatibility groups.

Another way to classify plasmids is by function. There are five main classes:

• Fertility-F-plasmids, which contain tra-genes. They are capable of

conjugation.
• Resistance-(R)plasmids, which contain genes that can build a resistance
against antibiotics or poisons. Historically known as R-factors, before the
nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production
of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances,
e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups.

Plasmids that exist only as one or a few copies in each bacterium are, upon cell
division, in danger of being lost in one of the segregating bacteria. Such single-
copy plasmids have systems which attempt to actively distribute a copy to both
daughter cells.

Some plasmids include an addiction system or "postsegregational killing system

(PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid
R1 in Escherichia coli.[2] They produce both a long-lived poison and a short-
lived antidote. Daughter cells that retain a copy of the plasmid survive, while a
daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate
because of the lingering poison from the parent cell.

Applications

Plasmids serve as important tools in genetics and biochemistry labs, where they
are commonly used to multiply (make many copies of) or express particular
genes.[3] Many plasmids are commercially available for such uses.

The gene to be replicated is inserted into copies of a plasmid which contains

genes that make cells resistant to particular antibiotics. Next, the plasmids are
inserted into bacteria by a process called transformation. Then, the bacteria are
exposed to the particular antibiotics. Only bacteria which take up copies of the
plasmid survive the antibiotic, since the plasmid makes them resistant. In
particular, the protecting genes are expressed (used to make a protein) and the
expressed protein breaks down the antibiotics. In this way the antibiotics act as a
filter to select only the modified bacteria. Now these bacteria can be grown in
large amounts, harvested and lysed (often using the alkaline lysis method) to
isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case,
researchers grow bacteria containing a plasmid harboring the gene of interest.
Just as the bacteria produces proteins to confer its antibiotic resistance, it can also
be induced to produce large amounts of proteins from the inserted gene. This is a
cheap and easy way of mass-producing a gene or the protein it then codes for, for
example, insulin or even antibiotics.

Plasmid DNA extraction

As alluded to above, plasmids are often used to purify a specific sequence, since
they can easily be purified away from the rest of the genome. For their use as
vectors, and for molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archetypes
of which are the miniprep and the maxiprep.[3] The former can be used to
quickly find out whether the plasmid is correct in any of several bacterial clones.
The yield is a small amount of impure plasmid DNA, which is sufficient for
analysis by restriction digest and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which
a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed
by additional purification. This results in relatively large amounts (several
micrograms) of very pure plasmid DNA.

In recent times many commercial kits have been created to perform plasmid
extraction at various scales, purity and levels of automation. Commercial services
can prepare plasmid DNA at quoted prices below $300/mg in milligram
quantities and $15/mg in gram quantities (early 2007).

Conformations

Plasmid DNA may appear in one of five conformations, which (for a given size)
run at different speeds in a gel during electrophoresis. The conformations are
listed below in order of electrophoretic mobility (speed for a given applied
voltage) from slowest to fastest:

• "Nicked Open-Circular" DNA has one strand cut.

• "Linear" DNA has free ends, either because both strands have been cut, or
because the DNA was linear in vivo. You can model this with an electrical
extension cord that is not plugged into itself.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has
been enzymatically "relaxed" (supercoils removed). You can model this by
letting a twisted extension cord unwind and relax and then plugging it into
itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with
both strands uncut, and with a twist built in, resulting in a compact form.
You can model this by twisting an extension cord and then plugging it into
itself.
 • "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired
regions that make it slightly less compact; this can result from excessive
alkalinity during plasmid preparation. You can model this by twisting a
badly frayed extension cord and then plugging it into itself.

The rate of migration for small linear fragments is directly proportional to the
voltage applied at low voltages. At higher voltages, larger fragments migrate at
continually increasing yet different rates. Therefore the resolution of a gel
decreases with increased voltage.

At a specified, low voltage, the migration rate of small linear DNA fragments is a
function of their length. Large linear fragments (over 20kb or so) migrate at a
certain fixed rate regardless of length. This is because the molecules 'reptate',
with the bulk of the molecule following the leading end through the gel matrix.
Restriction digests are frequently used to analyse purified plasmids. These
enzymes specifically break the DNA at certain short sequences. The resulting
linear fragments form 'bands' after gel electrophoresis. It is possible to purify
certain fragments by cutting the bands out of the gel and dissolving the gel to
release the DNA fragments.

Because of its tight conformation, supercoiled DNA migrates faster through a gel
than linear or open-circular DNA

Cloning vector

The pGEX-3x plasmid is a popular cloning vector.

A cloning vector is a small DNA vehicle that carries a foreign DNA fragment.
The insertion of the fragment into the cloning vector is carried out by treating the
vehicle and the foreign DNA with the same restriction enzyme, then ligating the
fragments together. There are many types of cloning vectors. Plasmids and
bacteriophages (such as phage λ) are perhaps most commonly used for this
purpose. Other types of cloning vectors include bacterial artificial chromosomes
(BACs) and yeast artificial chromosomes (YACs).

Common Features

Most commercial cloning vectors have a couple of key features that have made
their use in molecular biology so widespread.

Usually, the main purpose of cloning vector is the controlled expression of a

particular gene inside a convenient host organism (eg. E. coli). Control of
expression can be very important; it is usually desirable to insert the target DNA
into a site that is under the control of a particular promoter. Some commonly
used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower
mosaic virus's 35s promoter (for plant vectors). To allow for convenient and
favorable insertions, most cloning vectors have had nearly all their restriction
sites engineered out of them and a multiple cloning site (MCS) inserted that
contains many restriction sites. MCSs allow for insertions of DNA into the vector
to be targeted and possibly directed in a chosen orientation. A selectable marker,
such as antibiotic resistance [eg. beta-lactamase (see figure)] is often included in
the vector to identify positively transformed cells. All inserted DNA (plasmids
etc.) need an origin of replication (ORI; not shown in figure). High stringency
ORIs are preferable for cloning vectors.

Some other possible features of cloning vectors are: vir genes for plant
transformation, intergrase sites for chromosomal insertion, lac Z alpha fragment
for blue-white selection, and/or in-frame genes attached to the MCS for
recombinant proteins [eg. Green fluorescent protein (GFP) or glutathione S-
transferase (see figure)].

Screening: blue and white selection

General purpose vectors such as pUC19 usually include a system for detecting
the presence of a cloned DNA fragment, based on the loss of an easily scored
phenotype. The most widely used beta-galactosidase can be detected by its ability
to change the substrate X-gal (5 bromo-4-chloro-3-indolyl-beta-d-galactoside)
from colourless to blue. Cloning a fragment of DNA within the vector based gene
encoding the beta galactosidase prevents the formation of an active beta
galactosidase. If x gal is included in the selective agar plates, transformant
colonies are blue in the case of a vector with no inserted DNA and white in the
case of a vector containing a fragment of cloned DNA.