Defination And Introduction To The Transgenic

System:
Nowadays, breakthroughs in molecular biology
are happening at an unprecedented rate. One of
them is the ability to engineer transgenic
animals. The term "transgenics" refers to the
science of inserting a foreign gene into an
organism's genome. An animal is "transgenic"
once a scientist inserts DNA from another
organism. This process allows scientists to
transfer beneficial genes from a different animal,
bacterium, or plant. [6]
Defining The Term - Transgenic Animal:
There are various definitions for the term
transgenic animal.
A transgenic animal is one whose genome has
been changed to carry genes from other species.
[1]
Transgenic animals are animals which have been
genetically transformed by splicing and inserting
foreign animal or human genes into their
chromosomes.
The term transgenic animal refers to an animal
in which there has been a deliberate
modification of the genome - the material
responsible for inherited characteristics - in
contrast to spontaneous mutation. [2]
A transgenic animal is one which has been
genetically altered to have specific
characteristics it otherwise would not have. In
animals, transgenesis either means transferring
DNA into the animal or altering DNA already in
the animal.
Transgenic animals contain elements of two
different species - they are creatures that blur the
barrier between species.
The Federation of European Laboratory Animal
Associations defines the term as an animal in
which there has been a deliberate modification
of its genome, the genetic makeup of an
organism responsible for inherited
characteristics. [1]
An Introduction To The Technique:
The nucleus of all cells in every living organism
contains genes made up of DNA. These genes
store information that regulates how our bodies
form and function. Genes can be altered
artificially, so that some characteristics of an
animal are changed. For example, an embryo
can have an extra, functioning gene from
another source artificially introduced into it, or a
gene introduced which can knock out the
functioning of another particular gene in the

1
embryo. Animals that have their DNA
manipulated in this way are knows as transgenic
animals. [1]
Transgenic animals are produced by inserting
genes into embryos prior to birth. Each
transferred gene is assimilated by the genetic
material or chromosomes of the embryo and
subsequently can be expressed in all tissues of
the resulting animal. The objective is to produce
animals which possess the transferred gene in
their germ cells (sperm or ova). Such animals
are able to act as "founder" stock to produce
many offspring that carry a desirable gene or
genes. The animal that develops after receiving
the transgene DNA is referred to as the founder
(Fo) of a new transgenic lineage. If the germ
cells of the founder (mosaic or not) transmit the
transgene stably, then all descendants of this
animal are members of a unique transgenic
lineage.
A transgenic animal carries heterologous DNA
stably integrated into its genome. A transgenic
animal results from insertion of a foreign gene
into an embryo. The foreign gene becomes a
permanent part of the host animals' genetic
material. As the embryo develops, the foreign
gene may be present in many cells of the body,
including the germ cells of the testis or the
ovary. If the transgenic animal is fertile, the
inserted foreign gene (transgene) will be
inherited by future progeny. Thus, a transgenic
animal, once created, can persist into future
generations.
Transgenic animals are different from animals in
which foreign cells or foreign organs have been
engrafted. The progeny of engrafted animals do
not inherit the experimental change. The
progeny of transgenic animals do.
Why Transgenic Animals?
In some cases over expression of human genes
in bacteria (such as E. coli) does not yield a
protein that is functionally active in humans.
The reason for this is that some proteins need to
be post-translationally modified
(phosphorylated, glycosylated, etc.) before they
are active. Bacteria generally lack the specific
enzymes recognizing the human protein
sequences that need to be modified, and thus the
bacterially produced gene product will differ
from the native one. To counter this problem,
certain human genes can be introduced into farm
animals (usually yeast will do the job, too), and
when these genes are expressed in the mammary
glands of the animals, the post-translationally
2
modified protein can be isolated from milk,
tested whether its post-translationally modified
product is identical or at least very similar to the
native human one, and if so, be developed as a
pharmaceutical. For example, the genes for two
different human blood clotting factors (VIII and
IX) have been hooked up to sheep and pig
regulatory sequences that causes expression in
mammary tissue; after transformation of sheep
or pig embryos, genetically engineered animals
have been selected that produce milk with a
large percentage of human blood-clotting factor.
This protein can be isolated from the milk,
purified, and marketed. Similarly, transgenic
rabbits have been created that produce human
interleukin-2, which is a protein stimulating the
proliferation of T-lymphocytes; the latter play an
important role in fighting selected cancers. [3]
The majority of transgenic animals produced so
far are mice, the animal that pioneered the
technology. Long life cycles of farm animals
slow genetic analysis. That's why researchers
use smaller, faster-breeding animals such as
mice as model systems to test their ideas and
their DNA constructs. The first successful
transgenic animal was a mouse. Over 80% of
mouse genes function the same as those in
humans. Mice also have a short reproduction
cycle and their embryos are amenable to
manipulation. Mice are therefore an ideal human
surrogate in the study of most diseases.
Currently over 95% of transgenic animals used
in biomedical research are mice. Other
transgenic animals include rats, pigs and sheep.
It is hoped that the refinement of transgenesis
techniques in mice will ultimately allow for a
corresponding reduction in the use of "higher"
animals, such as dogs and non-human primates,
in biomedical research.A few years later; it was
followed by rabbits, pigs, sheep, fish, poultry
and cattle. Furthermore, the mouse is the only
model system that combines homologous
recombination with cloning to allow the study of
modified genes in development of adult animals.
Currently, in livestock homologous
recombination is possible only with cells grown
in tissue culture. This means a scientist can
study the effect of knocked-out genes only on
the physiology of the cell. The possible role of
the gene in development from embryo to adult
cannot be tested without a system of cloning:
taking the original cell and growing an adult
from it.

3
The insertion of a foreign gene (transgene) into
an animal is successful only if the gene is
inherited by offspring. The success rate for
transgenesis is very low and successful
transgenic animals need to be cloned or mated.
Since the early 1980s, methods have been
developed and refined to generate transgenic
animals or transgenic aquatic species. For
example, transgenic livestock and transgenic
aquatic species have been generated with
increased growth rates, enhanced lean muscle
mass, enhanced resistance to disease or
improved use of dietary phosphorous to lessen
the environmental impacts of animal manure.
Transgenic poultry, swine, goats, and cattle also
have been produced that generate large
quantities of human proteins in eggs, milk,
blood, or urine, with the goal of using these
products as human pharmaceuticals. Examples
of human pharmaceutical proteins include
enzymes, clotting factors, albumin, and
antibodies. The major factor limiting widespread
use of transgenic animals in agricultural
production systems is the relatively inefficient
rate (success rate less than 10 percent) of
production of transgenic animals.
Scientists do this, creating a "transgenic"
organism, to study the function of the introduced
gene and to identify genetic elements that
determine which tissue and at what stage of an
organism's development a gene is normally
turned on. Transgenic animals have also been
created to produce large quantities of useful
proteins and to model human disease. Various
human proteins that have been expressed in
transgenic animals include: anti-thrombin III (to
treat intravascular coagulation), collagen (to
treat burns and bone fractures), fibrinogen (used
for burns and after surgery), human fertility
hormones, human hemoglobin, human serum
albumin (for surgery, trauma, and burns),
lactoferrin (found in mother milk), tissue
plasminogen activator, and particular
monoclonal antibodies (including one that is
effective against a particular colon cancer).
Animals mostly used for this work are pigs,
cows, sheep, and goats.
Almost all the work on transgenic animals is still
at the research level. But it enjoys inherent
interest and the immense potential for future
commercial applications. Scientists, farmers and
business corporations hope that transgenic
techniques will allow more precise and cost-
effective animal and plant breeding programs.
4
They also hope to use these new methods to
produce animals with desirable characteristics
that are not available using current breeding
technology.
The technology has already produced transgenic
animals such as mice, rats, rabbits, pigs, sheep,
and cows. Although there are many ethical
issues surrounding transgenesis, this article
focuses on the basics of the technology and its
applications in agriculture, medicine, and
industry.
Historical Background
Prior to the development of molecular genetics,
the only way of studying the regulation and
function of mammalian genes was through the
observation of inherited characteristics or
spontaneous mutations. Long before Mendel and
any molecular genetic knowledge, selective
breeding was a common practice among farmers
for the enhancement of chosen traits, e.g.,
increased milk production. [2]
In the 1970s, experiments were conducted with
embryonal carcinoma cells and teratocarcinoma
cells to construct chimeric mice (Brinster, 1974;
Mintz and Illmensee, 1975; Bradley et al.,
1984). In these chimeric animals, cultured cells
derived from one strain of mouse were
introduced into the embryos of another strain of
mouse by direct embryo aggregation or by
injection into the blastocyst stage embryo. [2]
The mutual contributions of developmental
biology and genetic engineering permitted rapid
development of the techniques for the creation
of transgenic animals. DNA microinjection, the
first technique to prove successful in mammals,
was first applied to mice (Gordon and Ruddle,
1981) and then to various other species such as
rats, rabbits, sheep, pigs, birds, and fish. Two
other main techniques were then developed:
those of retrovirus-mediated transgenesis
(Jaenisch, 1976) and embryonic stem (ES) cell-
mediated gene transfer (Gossler et al., 1986). [2]
Since 1981, when the term transgenic was first
used by J.W. Gordon and F.H. Ruddle (1981),
there has been rapid development in the use of
genetically engineered animals as investigators
have found an increasing number of applications
for the technology. [2]
During 1982-83 pioneering research featured
transgenic mice given a copy of the gene for
human growth hormone. In this case, the added
gene was inserted randomly in the mouse
genome. It did not insert at the mouse gene for
growth hormone. The addition of the gene for
5
human growth hormone did not inactivate or
"knock-out" the genes for mouse growth
hormone. [4]

A picture of two mice side by side, one with the

extra gene and the other without, gave "visual
impact of what this technology might do,"
Pinkert pointed out. Comparing the size of the
mouse with the extra gene to the control was like
comparing a softball to a baseball. [4]
In 1997 the cloning of Dolly and engineering of
Polly have combined transgenesis and cloning.
This combination was an essential step in
developing in livestock a system of homologous
recombination to modify existing genes. [4]
Timeline Of Animal Cloning And Gene
Transfer:
·1891 first successful embryo transfer early
1900's in vitro embryo culture develops
· 1961 mouse embryo aggregation to produce
chimeras
· 1966 first report of microinjection of mouse
embryos
· 1973 foreign genes function after cell
transfection
· 1974 development of teratocarcinoma cell
transfer
· 1977 mRNA and DNA transferred to Xenopus
eggs
· 1980 mRNA transferred into mammalian ova
· 1980-81 transgenic mice first documented
· 1981 transfer of ES cells derived from mouse
embryos
· 1982 transgenic mice and a growth hormone
phenotype
· 1983 tissue specific gene expression in
transgenic mice
· 1985 transgenic domestic animals produced
· 1985 microinjection for transgenic pigs, sheep,
rabbits, fish
· 1987 chimeric "knock-out" mice described
· 1987 retrovirus mediated: transgenic chicken
· 1989 targeted DNA integration & germline
chimeric mice
· 1989 microinjection for transgenic cattle
( Russia )
· 1989 first sperm mediated reports in farm
animals

6
· 1991 microinjection for transgenic goats first
refereed publication
· 1993 germline chimeric mice produced using
co-culture
· 1996 ES cells used for nuclear transfer: sheep
· 1997 somatic cells from adult sheep used for
cloning by nuclear transfer (Dolly). [4]
Production Of Transgenic Animals:
Scientists can now produce transgenic animals
because, since Watson and Crick’s discovery (in
1953), there have been breakthroughs in:
1. Recombinant DNA (artificially-produced
DNA)
2. Genetic cloning
3. Analysis of gene expression (the process by
which a gene gives rise to a protein)
4. Genomic mapping [1]
Principle:
The underlying principle in the production of
transgenic animals is the introduction of a
foreign gene or genes into an animal (the
inserted genes are called transgenes). The
foreign genes “must be transmitted through the
germ line, so that every cell, including germ
cells, of the animal contains the same modified
genetic material.”(Germ cells are cells whose
function is to transmit genes to an organism’s
offspring.)[1]
This is a brief outline of the steps necessary to
obtain transgenic mice or rats: DNA is prepared
and microinjected into fertilized mouse or rat
eggs. Potentially transgenic rodents are born.
Transgenic founders are identified and bred to
produce offspring for analysis. Core personnel
are available for consultation on all aspects of
transgenic research. Much genetic engineering
goes into the choice of a foreign gene and
building a construct. The construct must have
promotes to turn on foreign gene expression at
its new site within the host animal genome. By
choosing a particular promoter and splicing it in
front of the foreign gene, we can encourage
expression of our transgene within a specific
tissue.
A transgenic animal for pharmaceutical
production should (1) produce the desired drug
at high levels without endangering its own
health and (2) pass its ability to produce the drug
at high levels to its offspring. The current
strategy to achieve these objectives is to couple
the DNA gene for the protein drug with a DNA
signal directing production in the mammary
gland. The new gene, while present in every cell
of the animal, functions only in the mammary
7
gland so the protein drug is made only in the
milk. Since the mammary gland and milk are
essentially "outside" the main life support
systems of the animal, there is virtually no
danger of disease or harm to the animal in
making the "foreign" protein drug. After the
DNA gene for the protein drug has been coupled
with the mammary directing signal, this DNA is
injected into fertilized cow, sheep, goat, or
mouse embryos with the aid of a very fine
needle, a tool called a micromanipulator, and a
microscope (Figure 2). The injected embryos are
then implanted into recipient surrogate mothers
where, hopefully, they survive and are born
normally. [14]

Production of Transgenic animal is entirely

described here:
1. Plan the experiment:
What is the purpose of your experiment? Do you
want to define tissue specific regulatory
sequences? Do you want to overexpress a
protein in a specific cell lineage? You will need
to obtain or clone the desired promoter and
structural gene. Expression of some genes will
be deleterious or incompatible with proper
growth and development of the embryo. Special
arrangements should be made if you expect
embryonic lethality from transgene expression.
The expression of a transgene requires that the
appropriate transcriptional control elements be
included in the DNA construct. A literature
search may identify these elements. Preliminary
studies in cell cultures are recommended to
verify the integrity of the construct and the
function of the promoter. However, it is not
always possible to predict in advance whether
the transgene will have the capability of being
expressed in vivo. A review of reporter
molecules is available. The Core has a nuclear
localized lacZ reporter vector for investigators

8
who wish to characterize regulatory elements in
transgenic mice. A completed materials transfer
agreement is required before this plasmid can be
distributed to investigators. Commercially
available vectors that may be useful in
transgenic research include: 1) the CMV-IE
promoter for widespread gene expression, 2)
tetracycline regulated gene expression systems
for inducible gene expression, and 3) luciferase
and green fluorescent protein reporter genes. [7]
2. Transgene DNA Preparation :
Although the basic coding system is the same in
all organisms, the fine details of gene control
often differ. A gene from a bacterium, say, will
often not work correctly if it is introduced
unmodified into a plant or animal cell. The
genetic engineer must first construct a transgene
– the gene to be introduced plus a control
sequence. All genes are controlled by a special
segment of DNA found on the chromosome next
to the gene and called a promoter sequence.
When
constructing a transgene, scientists generally
substitute the original promoter sequence with
one that will be active in the correct tissues of
the recipient animal or plant and which may also
allow them to turn the gene on or off as needed.
For example, a promoter sequence that requires
a dietary "trigger" substance can be used to turn
on a new hormone gene in animals; the animal
does not produce the new hormone unless fed
the appropriate trigger.
In order for a transgenic technique to work, the
genetic engineer must first construct a transgene,
which is the gene to be introduced plus a control
sequence. When making a transgene, scientists
usually substitute the original promoter sequence
with one that will be active in the correct tissues
of the recipient plant or animal. The gene for the
target protein is linked with a milk-specific
promoter. Simply put, the investigator constructs
a transgene with a promoter and a structural
gene for example a reporter gene such as lacZ or
a transcription factor. [9]
The transgene DNA is engineered in the
molecular laboratory to achieve fairly
predictable expression in the animal. Using
restriction enzymes and ligase, different
functional regions of genes from different
species may be recombined in the test tube. All
components of endogenous genes may be
isolated and recombined to form a transgene
expression cassette or construct. The ends of the
completed construct may be modified by the
9
addition of polylinker sequences containing
several different restriction enzyme recognition
sites. The polylinker permits the construct to be
inserted into a variety of vectors for testing and
cloning. The following review of endogenous
gene components will clarify these strategies:

(Figure 1. Schematic diagram of the double-

stranded DNA regions of the transgene
expression cassette. )
Restriction enzyme recognition sites are
clustered at either end of the cassette (i.e.,
upstream and downstream).
"ATG" indicates the beginning of the
transcriptional reading frame.
"SIG" indicates the signal sequence.
"AAA" indicates the poly-A tail [9]
The endogenous gene contains exons that code
for specific portions of the final protein and
introns that appear necessary for optimal
expression of the gene (Figure 2). The
endogenous gene is flanked by non-coding DNA
sequences that regulate gene expression.
(Regulatory elements may also reside within
intragenic intron sequences.) Sequences located
at the 5' end of the gene are known as upstream
elements, while downstream elements are found
past the 3' end of the gene sequence. Regulatory
elements called promoters are usually found
immediately upstream of the gene, and have
critical roles in the temporal and tissue-specific
regulation of gene expression. Other regulatory
elements called enhancers function to enhance
gene expression, independent of their location
and orientation with respect to the gene.
Enhancer regions appear to correlate with
DNAase hypersensitive sites and may be several
kilo bases (kb) distant from the gene. Signal
sequences are short sequences that target protein
synthesis into specific intracellular pathways and
frequently direct secretion of the protein from
the cell. Secretory signals usually are found
directly adjacent to the 5' end of the gene, and
organelle targeting sequences usually are found
within the 3' end of the gene or immediately
downstream of the 3' end. These signal
sequences are within the reading frame or
transcriptional region of the gene and therefore
encode mRNA and short polypeptide products.
The 3' end of the reading frame also must
contain a poly-A nucleotide sequence to ensure
proper mRNA transcription and translation. [9]

10
(Figure 2. Comparison of the two forms of
transgenes that may be introduced into
embryonic pronuclei.)
The genomic form includes all naturally-
occurring intron elements that are involved in
mRNA splicing and expression, whereas the
shorter cDNA form is a synthetic sequence
representing only the protein-encoding exon
elements of the gene. [9]
Steps of transgene preparation:
1) The gene of interest is isolated on a strand of
DNA.
2) DNA is cut at specific points by restriction
enzymes. The enzymes recognize certain
sequences of bases on the DNA strand and cut
where those sequences appear.
3) The cut DNA joins with a vector, which may
be a virus or part of a bacterial cell called a
plasmid. The vector carries the gene of interest
into the organism that will produce the protein.
4) Transformation occurs when the gene carried
by the vector is incorporated into the DNA of
another organism where it initiates the action
desired (production of a drug, etc.) [14]

3. Insertion of the transgene into animals :

By genetic engin

eering,
the DNA gene for a protein drug of interest can
be transferred into another organism that will
produce large amounts of the drug. This
technique can be used to impart new production
characteristics to an organism, as well as to
trigger the production of a protein drug. Copies
of the transgene are usually injected directly into
a fertilized egg which is then implanted in the
11
female productive tract. However, it is difficult
to control where in the chromosome the
transgene is inserted, and this sometimes causes
variations in the level at which the gene is
expressed. As well, the process is technically
demanding and has a low success rate. Currently
less than 5 per cent of injected embryos result in
offspring with the gene integrated into their
DNA and able to be passed on consistently to
successive generations. Researchers are
therefore investigating new methods of gene
transfer
Gene Transfer Methods:
1.Microinjection of cells (oocytes) with DNA
2.ES (embryonic stem) cell transfer
3.Retroviral infection
4.Blastomere/embryo aggregation
5.Teratocarcinoma cell transfer
6.Electrofusion
7.Nuclear transplantation
8.Sperm-mediated transfer
9.Particle bombardment ("gene gun")
To date, there are three basic methods are used
for producing transgenic animals:
· DNA microinjection
· Retrovirus-mediated gene transfer
· Embryonic stem cell-mediated gene transfer[1]
(A). DNA Microinjection:
Gene transfer by microinjection is the
predominant method used to produce transgenic
farm animals. Since the insertion of DNA results
in a random process, transgenic animals are
mated to ensure that their offspring acquire the
desired transgene. However, the success rate of
producing transgenic animals individually by
these methods is very low and it may be more
efficient to use cloning techniques to increase
their numbers. For example, gene transfer
studies revealed that only 0.6% of transgenic
pigs were born with a desired gene after 7,000
eggs were injected with a specific transgene.
The mouse was the first animal to undergo
successful gene transfer using DNA
microinjection. [1]

12
(Figure 3. Sequence of events in the generation
of a transgenic animal by pronuclear
microinjection) [1]
A. The double-stranded DNA components of the
transgene are combined enzymatically to yield a
transgene expression cassette.
B. Transgene cassettes are inserted into plasmid
vectors and cloned.
C. Transgene-bearing plasmids are transfected
into cultured eukaryotic cells to evaluate
expression of the transgene.
D. Plasmid-free transgene fragments are
introduced directly into embryonic pronuclei.
E. Manipulated embryos are placed in the
reproductive tract of a pseudopregnant recipient.
F. The genomic DNA of live-born pups is
analyzed for the presence of the transgene DNA
sequence. [9]

(Injection of cloned DNA into embryos: One cell

embryo is positioned for micro-injection into the
pronucleus (left). The plasma membrane has
been pierced, and the tip of the needle remains
inside the pronucleus, while DNA is expelled
from the needle, causing the pronucleus to swell
visibly.)
This method involves the direct microinjection
of a chosen gene construct (a single gene or a
combination of genes) from another member of
the same species or from a different species, into
the pronucleus of a fertilized ovum. It is one of
the first methods that proved to be effective in
mammals (Gordon and Ruddle, 1981). The
introduced DNA may lead to the over- or under-
expression of certain genes or to the expression
of genes entirely new to the animal species. The
insertion of DNA is, however, a random process,
and there is a high probability that the
introduced gene will not insert itself into a site
on the host DNA that will permit its expression.
The manipulated fertilized ovum is transferred
into the oviduct of a recipient female or foster
mother that has been induced to act as a
recipient by mating with a vasectomized male.
The first successful production of transgenic
mice using pronuclear microinjection was
reported in 1980 (Gordon et al., 1980)

13
Embryo Collection:
The choice of the donor parental strains for
production of the pronuclear embryos is a point
of extreme proprietary concern to most
laboratories. Many factors are cited including
the response to superovulation, frequencies of
embryo survival following microinjection, size
of pronuclei and the incidence of specific
pathologies inherent in various strains. The
relative merit of inbred versus outbred
backgrounds may be important for the
evaluation of a specific transgene expression.
Other factors may involve coat color, the
availability of a certain strain, or simply
anecdotal rationales [9]
On the morning following breeding, oviducts are
removed from euthanized donors, and clumps of
pronuclear embryos are collected from the
oviducts by flushing or by dissection into a
microdrop of sterilized buffered medium. The
embryos are clumped together with sticky
follicular cumulus cells that must be removed by
brief treatment in a series of microdrops. The
first drop, a solution of the enzyme
hyaluronidase, is followed by two or more wash
drops. Using heat-pulled tapered micropipettes
controlled by mouth suction (a new pipette for
each drop), the embryos are transferred from
drop to drop until they are free of cumulus cells,
debris and enzyme. Finally, the embryos are
transferred into a pool of medium in a petri dish
that will be placed under the microscope. The
embryo-containing pool is covered by a layer of
sterile-filtered, autoclaved mineral oil to prevent
contamination by microorganisms and debris
and to prohibit evaporation and the resultant pH
changes that would kill the embryos. All
collection and manipulation media contain a
buffering system (i.e., bicarbonate or HEPES)
and protein source (e.g., bovine serum albumin)
to prevent embryos from adhering to the dishes
and pipettes. In addition, media may contain
antibiotics (e.g., penicillin and/or streptomycin)
and a heavy-metal chelating agent (e.g., EDTA).
Equipment:
The equipment required to perform
microinjection can cost between $50,000 and
$80,000 and includes:
·CO2 incubator to maintain manipulated
embryos at 37-38° C in an atmosphere of 5-6
percent CO2.
·Inverted microscope with a fixed stage.
·Phase contrast, Nomarski differential
interference, or Hoffman modulated contrast
14
optical systems to visualize pronuclei. With 10x
or 15x eyepieces, a 20x or 40 x objectives is
required.
· A pair of micromanipulators to control the
DNA injection pipette and the embryo-holding
pipette.
· A pair of micro-volume syringes and associated
tubing to regulate the fluid dynamics in the
injection and holding pipettes. (Expensive
automatic microinjection systems are available
in lieu of the injection syringe.)
· Pipette-pulling apparatus.
· Vibration-free pneumatic table (optional).
· Microforge apparatus to heat-polish and bend
pipets.
· Pipette bevelling apparatus (optional).
· Supply of clean capillary pipettes for the
manufacture of holding and injection pipettes.
· Fluorinert solution (optional) to provide
optimal fluid dynamics in the pipettes.
· Microphotographic equipment (optional)
including 35mm camera and/or video recording
apparatus. [9]
The petri dish containing the embryo microdrop
is placed into focus at a relatively low
magnification, and degenerated embryos may be
culled from the healthy embryos at this time.
The holding pipet is brought down into the
medium, and the first embryo is gently sucked
onto the end of the pipet and held in place. The
tip of the injection pipet is brought into the same
plane of focus as the pronucleus to be injected,
and a small amount of DNA solution is ejected
to ensure the patency of the pipet. The injection
pipet is then thrust through the zona pellucida,
cell membrane, cytoplasm and nuclear
membrane in a single smooth motion. Even if
the membrane appears to have been pierced, the
only reliable indication of success is the swelling
of the pronucleus (volume = approximately 1
pl). The pipet is removed smoothly, and the
injected embryo is moved to the far end of the
pool of medium before the next is processed.
Once a group of embryos has been completed, it
is transferred in a single volume of medium to
another dish for incubation and visual evaluation
within a few hours. All apparently viable
embryos are then transferred to a recipient
female oviduct.
Embryo Transfer:
The manipulated embryos must be transferred
into a suitable reproductive tract in order to have
an opportunity to become live-born transgenic
mice. The recipient female optimally should be
15
somewhat earlier in her reproductive cycle than
the embryo donor because manipulated and
cultured embryos exhibit slightly retarded
development when compared to embryos that
developed in vivo. Recipients for embryo
transfer are prepared by mating with
vasectomized males at the same time that the
superovulated donor females are mated with
fertile males. Recipient females are anesthetized,
the skin and peritoneum are incised, and the
ovarian fat pad and bursa are exteriorized and
draped over the midline. The bursa is opened,
avoiding any prominent vessels, and the
infundibulum is located. An embryo transfer
pipet with an internal diameter of less than 150
µm is loaded in the following sequence: one
small air bubble, approximately 10 µl of
medium, a second air bubble, 2-15 embryos in
less than 25 µm of medium, and a third air
bubble. The pipet tip is inserted into the
infundibulum of the oviduct, and the contents
are gently transferred into the oviduct by mouth
pressure until the middle air bubble is expelled.
The reproductive tract is gently replaced and the
incision is closed. Pregnancy should be visible
about two weeks after the embryo transfer (post-
ET), and the litter should be delivered about
three weeks post-ET. Animals may be analyzed
for the presence of the transgene in their
genomes after weaning at six weeks post-ET
(Figure 3F). It should be noted that certain
transgene sequences may be activated in uterus
and may affect embryo survival or gestation
length. Also, transgenic females in subsequent
generations should be observed for abnormal
gestation lengths.
A major advantage of this method is its
applicability to a wide variety of species.
(B). Embryonic stem cell-mediated gene
transfer:
This method involves:
· Isolation of totipotent stem cells (stem cells
that can develop into any type of specialized
cell) from embryos
· The desired gene is inserted into these cells.
· Cells containing the desired DNA are
incorporated into the host’s embryo, resulting in
a chimeric animal. [1]
Unlike the other two methods, which require
live transgenic offspring to test for the presence
of the desired transgene, this method allows
testing for transgenes at the cell stage. [1]
This method involves prior insertion of the
desired DNA sequence by homologous
16
recombination into an in vitro culture of
embryonic stem (ES) cells. Stem cells are
undifferentiated cells that have the potential to
differentiate into any type of cell (somatic and
germ cells) and therefore to give rise to a
complete organism. These cells are then
incorporated into an embryo at the blastocyst
stage of development. The result is a chimeric
animal. ES cell-mediated gene transfer is the
method of choice for gene inactivation, the so-
called knock-out method. [2]
This technique is of particular importance for the
study of the genetic control of developmental
processes. This technique works particularly
well in mice. It has the advantage of allowing
precise targeting of defined mutations in the
gene via homologous recombination. [2]
(C). Retrovirus-mediated gene transfer.
The third method produces chimeras, altered
animals with mixed DNA. To increase the
probability of expression, gene transfer is
mediated by means of a carrier or vector,
generally a virus or a plasmid. Retroviruses are
commonly used as vectors to transfer genetic
material into the cell, taking advantage of their
ability to infect host cells in this way. Offspring
derived from this method are chimeric, i.e., not
all cells carry the retrovirus. Transmission of the
transgene is possible only if the retrovirus
integrates into some of the germ cells. [2]
A retrovirus is a virus that carries its genetic
material in the form of RNA rather than DNA.
This method involves:
1. Retroviruses used as vectors to transfer
genetic material into the host cell, resulting in a
chimera, an organism consisting of tissues or
parts of diverse genetic constitution.
2. Chimeras are inbred for as many as 20
generations until homozygous (carrying the
desired transgene in every cell) transgenic
offspring are born
The method was successfully used in 1974 when
a simian virus was inserted into mice embryos,
resulting in mice carrying this DNA.
4. Selection of Gene Targeted Cells :
Homologous recombination is a very rare event,
and scientists using it to modify or "knock out"
mouse genes must identify the cells in which it
has occurred. In addition to injecting the gene
they are trying to incorporate, scientists also
inject "selectable" genes whose products permit
cells to live or cause them to die in the presence
of a particular drug. The two most common
selectable genes used in gene targeting are the
17
neomycin resistance (neor) gene, which allows
cells to survive in the presence of the antibiotic
neomycin, G418, and the thymidine kinase (TK)
gene from the herpes virus. Cells with this gene
die in the presence of the antiviral agent
gancyclovir. The neor and TK genes are
generally used together for maximum selection.
[12, 15]

Scoreable vs. Selectable Markers:

Scoreable markers are an example of genes that
are easy to find. Examples include a gene that
makes an enzyme that makes a colour (such as
GUS) or that makes light (such as lux genes for
luciferase) or that makes a fluorescent protein
(such as the gene for Green Fluorescent Protein).
The two genes the 'gene of interest' and the
'selectable marker' are attached ('in tandem'). So
where the scoreable marker goes, it is very likely
the gene of interest goes too.
Another example of a gene that is easy to find is
a gene for resistance to an antibiotic. This is
called a selectable marker. These are even more
powerful than scoreable markers. Cells that
receive a copy of the gene for resistance to the
antibiotic kanamycin can grow in test tubes
containing kanamycin, while other cells without
the gene will die.
With a scoreable marker, you may be able to
pick out the one fluorescent cell in a thousand
other cells that don’t fluoresce. But with a
selectable marker, only the cells with the marker
remain alive after treating with the antibiotic. It
is the difference between an orange vest to make
a cell stand out in a crowd and a bullet-proof
vest that lets a cell survive the attack of
antibiotics. [12]
Now you have your gene of interest and you can
follow it using the attached selectable marker.
5. Clone and the Verify the Integrity of the
Transgene :

18
In transgene design several things should be
considered during cloning. For exempt,
prokaryotic vector sequences interfere with the
expression of some transgenes, thus unique
restriction sites at the 5' and 3' ends of the
construct should be available for vector removal.
The transgene should contain unique markers so
that its presence can be easily detected in DNA
samples and so that its expression can be
assayed and distinguished from endogenous
gene expression. Sequencing of junction
fragments should be carried out in order to
confirm that the transgene has a functional
promoter, initiation codon, and polyadenylation
signal. Under the best circumstances, the
transgene is tested for expression in a tissue
culture system before transgenic mice are made.
[7]
6. DNA Purification and MICROINJECTION
7. Establish a Screening Method :
A PCR assays should be established to rapidly
identify transgenic animals. A second assay that
will detect an endogenous mouse gene, such as
beta-globin, or an endogenous rat gene, such as
prolactin, is required in order to demonstrate that
the DNA preparations are amenable to PCR.
Animals are tested with both assays so that no
transgenic founder is mistakenly discarded
because the tail DNA is not suitable for PCR. A
Southern blot assay is also needed. [7]
8. Establish an Expression Assay :
It's important to show that transgene is
expressed. RNA expression can be detected by
in situ hybridization or RNAse protection assay
with RNA probes. Alternatively, an RT-PCR
approach can be used.. [7]
9.Screen Potential Founders :
A genetic founder, denoted "F0," is a first-
generation transgenic animal that develops
directly from a microinjected embryo and carries
the transgene stably integrated in its genome.
Transgene sequences are analyzed by
biochemical and molecular methods to confirm
that they are complete and intact. If they are,
then the animals are raised to maturity and bred
to non-transgenic mates. Their offspring are
tested to confirm that the transgene is inherited
at the expected frequency.
Milk is collected from these transgenic animals
and analyzed to measure the expression levels
and biochemical characteristics of the
recombinant protein. Milk may be collected
directly from female founders, or from the
daughters of male founders.
19
Tail biopsies from potentially transgenic animals
will be obtained 5 weeks after injecting eggs (3
weeks gestation time and 2 weeks of post-natal
growth). DNA is extracted from the tail biopsies
by simple salt out methods or you can use kits
from various vendors on the market. For speed,
some people use the "HotShot" method. Once
the investigator has the DNA we expect you to
test each DNA sample for both the transgene and
an endogenous mouse gene or rat gene by PCR.
Ideally, you will identify which pups are
transgenic before they are weaned at three weeks
of age so that only transgenic pups are moved to
your animal room. If the testing is not complete
then we will transfer all of the pups to your
animal room. [7]
10 . Breeding and Analysis of Transgenic
Rodents :
The final stage in the process is to study animals
carrying the transgene. Typically, the transgenic
founder animals are bred to mice of defined
genetic background such as C57BL/6.
Transgenic rats are bred to Sprague-Dawley
since that is their originating genetic
background. Analysis of transgene expression
and the consequences of expression are
generally conducted in the offspring. The best
strategy, if applicable is transgenic founder
analysis. This eliminates the time and cost of
breeding multiple offspring from each founder.
[7]
Examples Of Transgenic Animals:
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.
Many more animal clones have been generated
in the mean time. For example, cloned cows
appeared in 1999 and now there are cloned pigs
that have been modified to reduce transplant
rejection of pig organs in humans. Cloned pets
(cats and dogs) have been created too. There are
even cloned mules.
Harvard scientists made a major scientific
breakthrough when they received a U.S. patent
(the company DuPont holds exclusive rights to
its use) for a genetically engineered mouse,
20
called OncoMouse® or the Harvard mouse,
carrying a gene that promotes the development
of various human cancers. [1]
GSK scientists engineered the overexpression of
the human mitochondrial transporter protein,
"uncoupling protein-3" (UCP-3), in skeletal
muscle in mice. In this model, the transgenic
mice were found to eat more than wild-type
littermates, yet remain leaner and lighter. The
mice also exhibit lower glucose and insulin
levels and an increased glucose clearance rate,
leading to the hypothesis that compounds that
regulate expression of UCP-3 might be of use in
treating obesity. [5]
In theory, large quantities of the human protein
can be produced in the animal's milk and
subsequently purified for use in medical
therapies. It has been gaining application among
biotechnologists since the development of
transgenic "super mice" in 1982 and the
development of the first mice to produce a
human drug, tPA (tissue plasminogen activator
to treat blood clots), in 1987.
In 1997, the first transgenic cow, Rosie,
produced human protein-enriched milk at 2.4
grams per litre. This transgenic milk is a more
nutritionally balanced product than natural
bovine milk and could be given to babies or the
elderly with special nutritional or digestive
needs. Rosie’s milk contains the human gene
alpha-lactalbumin. [1]
The A. I. Virtanen Institute in Finland produced
a calf with a gene that makes the substance that
promotes the growth of red cells in humans. [1]
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. [1]
Advantages:
The benefits of transgenic animals include:
· Large-scale, low-cost production independent
of proximity to the oceans and with reduced
environmental impacts;
·Increased growth rates;
·Improved disease resistance;
·Improved food-conversion rates;
·Leaner meat;
21
·Increased muscle mass;
·Improved wool quality;
·Improved nutritional quality or appeal; and
·More efficient use of indoor water-recycling
plants.
Specifically, the environmental and nutritional
benefits resulting from aquaculture outweigh
potential risks, which include the runoff of
pollutants into closed or semi-closed waterways;
the elimination of coastal forests and
ecosystems; the potential for increases in
waterborne disease and parasites; and
sustainability. [8]
Advantages Over Selective Breeding:
Transgenic technology is an extension of
agricultural practices that have been used for
centuries: selective breeding and special feeding
or fertilizing programs. It may reduce or even
replace the large-scale use of pesticides and
long-lasting herbicides. Transgenic technology is
still experimental and is still very expensive.
However, it offers a number of advantages over
traditional methods.
Compared with traditional methods, transgenic
breeding is:
More specific – scientists can choose with
greater accuracy the trait they want to establish.
The number of additional unwanted traits can be
kept to a minimum.
Faster – establishing the trait takes only one
generation compared with the many generations
often needed for traditional selective breeding,
where much is left to chance.
More flexible – traits that would otherwise be
unavailable in some animals or plants may be
achievable using transgenic methods. Less
costly – much of the cost and labour involved in
administering feed supplements and chemical
treatments to animals and crops could be
avoided.
Environmentally friendly – allowing less use of
chemical pesticides and herbicides and reduced
tillage leading to less land degradation.
Overall, the use of transgenic technology has
many advantages over traditional methods.
Transgenic breeding is said to be more specific,
faster, and less costly. Right now research is
limited to traits involving one or a few genes.
Before scientists can manipulate complex traits,
there is going to be the need for many years of
research.
Applications Of The Transgenic Animal
Research into transgenic animals could prove
useful in several ways. Scientists can provide
22
animals with beneficial genes or traits, such as
disease resistance, that will improve their quality
of life and bolster waning populations.
Transgenic animals may also be designed for
organ production, helping to ease the critical
shortage of kidneys and livers available for
transplants. In addition, scientists are
researching ways to produce proteins or drugs in
transgenic animals.
Applications of transgenic animals are described
in detail in this section, which can be
categorized in three groups:
Medicinal Applications:
1. Models of human disease processes:
One of the most important applications of
transgenic animals is the development of new
animal models for human disease. Gene
targeting is being exploited by scientists to
create models of human disease. The genetic
setup of an animal may be modified in such a
way that it develops a disease similar to an
equivalent human disease.
Hundreds of transgenic rodent lines have been
produced by introducing into the genome
genetic sequences such as viral transactivating
genes and activated oncogenes implicated in
specific pathologies. Transgenic rodent models
have been characterized for several human
diseases including cardio-vascular disease
(Walsh et al., 1990), cancer (Sinn et al., 1987),
autoimmune disease (Hammer et al., 1990),
AIDS (Vogel et al., 1988), sickle cell anemia
(Ryan et al., 1990), muscular dystrophy, Lou
Gehring’s disease, and neurological disease.
Here are some examples of transgenic animals
developed as models of disease: Human
Transgenic animals can serve as models for
many malignant tumors. Inserting the c-myc
oncogene, which regulates cell growth, into a
mouse creates a transgenic strain with a high
rate of spontaneous tumors. The type of tumor
depends on the promoter placed in front of the c-
myc gene in the contruct. The mammary tumor
virus (MTV) promotor increases the incidence
of breast adenocarcinomas. The immunoglobulin
heavy-chain enhancer (IgH), when inserted
along with the c-myc, results in a strain of mice
with a high incidence of lymphoblastic
lymphomas. Although mice have been the most
frequent hosts for transgenic modification, other
domestic animals have also been used.
Transgenic mice overexpressing the amyloid
precursor protein form deposits in the brain that
resemble the amyloid plaques found in
23
Alzheimer's patients. Mouse models such as
these can potentially be used to test drug
therapies and to learn more about the
progression of the disease.
Harvard scientists made a major scientific
breakthrough when they received a U.S. patent
(the company DuPont holds exclusive rights to
its use) for a genetically engineered mouse,
called OncoMouse® or the Harvard mouse,
carrying a gene that promotes the development
of various human cancers. [1]
Transgenic animals enable scientists to
understand the role of genes in specific diseases.
By either introducing or inactivating particular
genes, researchers can often for the first time
discover the root causes of diseases associated
with gene defects. For example, GSK scientists
engineered the overexpression of the human
mitochondrial transporter protein, "uncoupling
protein-3" (UCP-3), in skeletal muscle in mice.
In this model, the transgenic mice were found to
eat more than wild-type littermates, yet remain
leaner and lighter. The mice also exhibit lower
glucose and insulin levels and an increased
glucose clearance rate, leading to the hypothesis
that compounds that regulate expression of
UCP-3 might be of use in treating obesity. [5]
Reasons for using the transgenic animal as a
model for human diseases:
Transgenics may spare the use of higher
animals. The creation of transgenic animals is
resulting in a shift from the use of higher order
species to lower order species. In the long term,
a reduction in the number of animals used, for
example to study human diseases is possible due
to a greater specificity of the transgenic models
developed. This shift in the patterns of animal
use is being monitored by the CCAC through the
use of the Animal Use Data Form. An example
of the replacement of higher species by lower
species is the possibility to develop disease
models in mice rather than using dogs or non-
human primates. [2] On the other hand, the
success of the method has led to using its
potential for investigating a wider range of
diseases and conditions. The actual use of some
species may be increased. [5]
2.As organ transplant donors to humans:
Another rapidly-moving field is the potential use
of transgenic pigs for use as organ transplant
donors to humans. Patients die every year for
lack of a replacement heart, liver, or kidney. For
example, about 5,000 organs are needed each
year in the United Kingdom alone. Transgenic
24
pigs may provide the transplant organs needed to
alleviate the shortfall. Currently,
xenotransplantation is hampered by a pig protein
that can cause donor rejection but research is
underway to remove the pig protein and replace
it with a human protein. [1]This kind of research
is still very much in its infancy. If successful,
however, this research could transform the lives
of the many patients awaiting organ transplants.
Transgenic animals are being developed by
some companies to provide new organs for
transplantation such as kidneys, livers and
hearts. Transgenic pigs with human histo-
compatibility genes have been bred in the hope
that their "humanized" organs will not be
rejected by a patient's immune system. [10]
3. Proteins of medical importance to humans:
One important application of transgenic
technology is the generation of transgenic
livestock as "bioreactors." Transgenic animals
can produce biological products. It may be
possible to use transgenic animals to make rare
biological products for medical treatment. Milk-
producing transgenic animals are especially
useful for medicines. Key human genes have
been introduced into sheep, cows, goats, and
pigs so that the human protein is secreted into
the milk of the transgenic animal. In theory,
large quantities of the human protein can be
produced in the animal's milk and subsequently
purified for use in medical therapies. [15]
Principle behind the technique:
The major function of the mammary gland is to
produce proteins. The mammary gland is
capable of producing milk that carries over
40g/L of protein. Advantage of the unique
properties of this "natural protein secretion
organ" is taken in this technique. By utilizing
molecular biology technology, we can design
DNA constructs that reliably express high levels
of therapeutic proteins in the milk of the animals
that carry the transgene. Advantage of the
normal mammalian protein processing
mechanisms is taken to synthesize properly
folded and assembled complex proteins.
Although the epithelial cells in the mammary
gland do not usually express antibodies, it has
been found that the machinery needed to
properly fold and assemble the heavy and light
chains of antibodies are well represented in these
cells. By utilizing the milk specific promoters to
express the heavy and light chains, the cellular
machinery is capable of secreting high levels of
properly folded antibody.
25
Since this is a mammalian cell system, it is
capable of post-translational modifications such
as glycosylation and gamma carboxylation.
Many recombinant proteins, most of which are
of human origin, require glycosylation for
proper function or pharmokinetics. This system
provides high level expression combined with
mammalian modifications-unique to production
systems. This method permits flexible scale-up
of protein manufacturing to meet increasing
production needs throughout the product
development process. Scale-up is as simple as
breeding more transgenic animals. This is easier
and less expensive than building and validating a
larger biopharmaceutical fermentation or
mammalian cell culture facility, therefore
reducing overall capital costs.
Examples:
An early example of this technology by John
Clark and colleagues was the production of
transgenic sheep expressing the human blood-
clotting factor IX needed by many patients with
hemophilia. These researchers placed the human
factor IX gene under the control of a piece of
sheep DNA that normally turns on the beta-
lactoglobulin gene in the mammary tissue.
Though the sheep secreted factor IX into their
milk, the levels of the protein were very small.
With advances in the efficiency of creating and
expressing genes in transgenic farm animals,
therapeutic proteins can now be isolated. [15]
In 1997, the first transgenic cow, Rosie,
produced human protein-enriched milk at 2.4
grams per litre. This transgenic milk is a more
nutritionally balanced product than natural
bovine milk and could be given to babies or the
elderly with special nutritional or digestive
needs. Rosie’s milk contains the human gene
alpha-lactalbumin. [1]
Human alpha-1-antitrypsin, a protein used to
treat the rare genetic disorder of alpha-1-
antitrypsin deficiency, is also produced by this
technique. [1]
Other human proteins that have been expressed
in transgenic animals include: anti-thrombin III
(to treat intravascular coagulation), collagen (to
treat burns and bone fractures), fibrinogen (used
for burns and after surgery), human fertility
hormones, human hemoglobin, human serum
albumin (for surgery, trauma, and burns),
lactoferrin (found in mother milk), tissue
plasminogen activator, and particular
monoclonal antibodies (including one that is
effective against a particular colon cancer).
26
Animals mostly used for this work are pigs,
cows, sheep, and goats. [3]
Comparison with other methods of protein
production:
There are four other means of commercial
protein production. E. coli production, which
was the first commercialized, is very efficient,
but limited to simple non-glycosylated proteins.
Although the cost of production is low, the cost
of processing and refolding the proteins is
significant.
Fungal systems, such as Pichia or filamentous
fungi allow efficient production of some
secreted proteins, but the glycosylation is
usually high mannose which can affect the
pharmokinetics of the protein.
There is also the baculovirus production system,
which can produce a wide range of proteins in
small scale, but has yet to be scaled up to
commercial levels.
The standard method for producing complex
glycosylated proteins, (i.e. Monoclonal
Antibodies) is with cell tissue culture. The
protein may be properly folded and modified,
but the low yields per cost of production facility
limit the number of proteins that can be
developed.
Recombinant protein concentrations in the milk
of transgenic animals are substantially higher
than levels attained in cell tissue cultures.
Expression levels of 2 to 10 grams of
recombinant protein per liter of milk are readily
achievable in transgenic livestock. In
comparison, highly optimized cell cultures can
typically generate 0.2 to 1 gram per liter of
culture medium. It appears that transgenic
technology can achieve the high levels of
recombinant protein production normally found
only in prokaryotic systems. It has the added
benefit in that it is a mammalian system that can
secrete complex, glycosylated proteins, similar
to tissue culture. Thus it has the best of both
technologies, with the added advantage of lower
capital cost for the production facility.
Transgenic production takes advantage of
normal mammalian protein processing
mechanisms to synthesize properly folded and
assembled complex proteins -- all within the
cells of the mammary gland. This method
permits flexible scale-up of protein
manufacturing to meet increasing production
needs throughout the product development
process. Scale-up is as simple as breeding more
transgenic animals. This is easier and less
27
expensive than building and validating a larger
biopharmaceutical fermentation or mammalian
cell culture facility, therefore reducing overall
capital costs.
Recombinant protein concentrations in the milk
of transgenic animals are substantially higher
than levels attained in cultures of yeast, bacteria,
insect cells or mammalian cells. Expression
levels of 2 to 10 grams of recombinant protein
per liter of milk are readily achievable in
transgenic livestock. In comparison, highly
optimized cell cultures can typically generate 0.2
to 1 gram per liter of culture medium.
Animal most commonly used for the purpose of
protein production:
In choosing a species of animal it is optimal to
have animals that have been bred for significant
milk production and also have a relative short
generation time. Formally, choices range from
mice, one of the model systems with a
generation time of 3 months and milk yield of 1
ml, to rabbits with an 8 month generation time
and 4 liter yield, to the largest commercial
species, the cow. Cows have a generation time
of 3 years, with an annual milk yield of 8000
liters.
Since time is critical, goats are a logical
alternative with a generation time of 18 months
and a yield of nearly 800 liters. As a dairy breed,
goats show efficiency of milk production that is
unrivaled. As a dairy production animal goats
are utilized all over the world. Significant
expression, (2-10g/L) of recombinant proteins in
lactating goats has been shown. With an annual
yield of 800 liters, over 1 kilogram of
recombinant protein can be produced per
lactating animal. The scale-up of the goat herd
following standard breeding is straight-forward
and the production of 100's of kilograms of
recombinant proteins can be readily achieved.
This is well within the levels expected for most
recombinant protein markets. Their dairy
characteristics combined with their relative short
generation time allow meeting our goals. Small
amounts of the recombinant protein for initial
testing can be delivered within a year, followed
by high levels during normal lactation. By
utilizing a known dairy animal, the scale-up for
large volume production is straightforward.
Goats are an ideal dairy species as produce large
volumes of milk with high protein content, and
are generally accepted as a source of dietary
milk. They are relatively easy to breed and
maintain. Goat milk has been extensively
28
characterized biochemically, and this makes it
more straightforward to develop protein
purification procedures.
4. To test the safety of new medicines and
vaccines:
Because transgenic models can highlight
specific characteristics such as certain
mechanisms involved in the formation of
tumors, they can demonstrate more clearly the
possible side effects of new therapies. Their use
in early toxicity trials may also serve to prevent
the subsequent use of a larger number of animals
in the development phase. Toxicity-sensitive
transgenic animals have been produced for
chemical safety testing.
Transgenic animals can also be used to test the
identity and purity of human proteins used as
drugs. A transgenic animal that makes a human
protein (e g human insulin) will recognise this
substance as its own and will therefore not
produce an immune response against it. As a
consequence, the identity and purity of the
product can be tested more efficiently in such
animals, thereby saving the use of many
laboratory animals otherwise needed to obtain a
statistically significant result. [13]
5. Genatic Research
Genetic models to study the effects of genetic
changes on development:
Frequently used in genetic research are
transgenic fruit flies (Drosophila melanogaster)
as genetic models to study the effects of genetic
changes on development. Flies are often
preferred over other animals for ease of culture,
and also because the fly genome is somewhat
simpler than that of vertebrates. Transgenic mice
are often used to study cellular and tissue
specific responses to the disease. [15]
Agricultural Applications:
6. Disease resistance:
Scientists are attempting to produce disease-
resistant animals, such as influenza-resistant
pigs, but a very limited number of genes are
currently known to be responsible for resistance
to diseases in farm animals. [1]
7. Production of milk low in cholesterol:
Transgenic cows exist that produce more milk or
milk with less lactose or cholesterol. [1]
8. The polled (hornless) condition in cattle. [10]
Industrial Applications:
9. material fabrication:
In 2001, two scientists at Nexia Biotechnologies
in Canada spliced spider genes into the cells of
lactating goats. The goats began to manufacture
29
 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. [1]
10.Increased Meat Production:

An abnormally high quantity of growth hormone

in the transgenic animal is responsible for
increased meat production. Pigs and cattle that
have more meat on them can be produced. In the
past, farmers used growth hormones to spur the
development of animals but this technique was
problematic, especially since residue of the
hormones remained in the animal product. [1]
11. Sheep that grow more wool:
Breeding transgenic sheep that grow better wool
without needing dietary supplements of sulphur-
containing amino acids is under research.
The Future:
Research is presently limited to traits involving
one or a few genes. It will probably require
many years of research before scientists can
manipulate complex traits (such as meat quality
or animal behaviour) that are influenced by
many genes.
Much current research focuses on the
understanding and developing useful promoter
sequences to control transgenes and establishing
more precise ways to insert and place the
transgene in the recipient. Much still needs to be
done to improve our knowledge of specific
genes and their actions and of the potential side
effects of adding foreign DNA and of
manipulating genes within an organism.
The role of transgenic animals in biomedical
research
1. The issue
2. The importance of animal research
3. What is a transgenic animal?
4. GSK's use of transgenics
5. The human genome project and the role of
transgenics
6. The role of transgenic animals

30
7. Some perceptions around transgenic animal
experiments
8. Conclusion

The issue
Transgenic (or genetically modified) animals are
proving ever more vital in the discovery and
development of new treatments and cures for
many serious diseases by helping scientists to
characterise the newly-sequenced human
genome. Without them, the pharmaceutical
industry's ability to discover new treatments
would be significantly reduced.
However, the development and use of transgenic
animals understandably raise a number of
concerns, and the term "genetically modified"
needs to be properly explained. This paper
therefore seeks to explain the multiple roles of
transgenic animals in biomedical research,
considers the current usage levels and discusses
the issues surrounding transgenic animals in
general.
Public policy position
← Transgenic animals are proving ever more
vital in helping to discover and develop new
treatments and cures for disease.
← Developments with transgenesis in mice will
contribute to the further reduction in the use
of "higher" animals, such as dogs and non-
human primates, in biomedical research.
← The animal welfare issues associated with
the use of transgenic animals are
fundamentally no different from those
associated with other animals in biomedical
research. It is the minimisation of any pain
or distress to individual animals in medical
research that is important, not the manner in
which the animals are bred.
← The development and subsequent use of
transgenic animals is subject to stringent
internal GSK review and government
regulations and oversight. Regulations that
control animal research permit the use of
animals only when no alternative exists.
GSK recognises that there is public concern
regarding transgenic animals and is committed
to addressing these concerns.
1. The importance of animal research
A major part of biomedical research is aimed at
understanding human biology at the cellular and
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molecular level in health and disease. Whereas
certain human characteristics are shared by no
other species or only by primates, there are
many other characteristics that are shared with
many species of animals. The use of animals has
therefore become fundamental to all aspects of
modern biomedical research from the study of
basic biological mechanisms, to the
understanding of disease pathology and the
development of new medicines for both human
and veterinary use.
Virtually every medical breakthrough in the 20th
century came about as a result of research with
animals and many more treatments and cures
still await discovery. The list is almost endless
but includes the discovery of insulin in the
1920s (research involving dogs), the successful
polio vaccine developed in the 1950s (research
involving monkeys), and the prevention of
measles during the 1970s (research involving
monkeys).
(Specific information concerning GSK's use of
animals in research can be accessed on
http://www.gsk.com/research/about/about_anim
als-care.html)
2. What is a transgenic animal?
A transgenic animal is one which has been
genetically altered to have specific
characteristics it otherwise would not have. In
animals, transgenesis either means transferring
DNA into the animal or altering DNA already in
the animal.
The earliest transgenic approaches involved
transferring DNA, usually by injection into a
fertilised mouse egg. However, since it is not
possible to control the site of integration of the
foreign DNA using this technique, it is a
relatively imprecise tool. Mice resulting from
this technique are generally called
"overexpressors".
A more direct way to determine the function of a
new gene is to alter the gene and then observe
the physical manifestation (phenotype) of that
genetic trait e.g., is the mouse obese, diabetic.
These types of mice are often called knock-outs
and for several years now, it has been possible to
generate "knock-out" mice which carry
specifically defined mutations in the gene of
interest using gene targeting in mouse
embryonic stem cells.

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In conventional gene knockout and
overexpression approaches, the genetic
modification is present in the animal at birth.
However, new molecular genetic techniques
have been developed that allow a particular gene
(in some cases in a specific organ or tissue) to be
switched on or off for a short period of time in
the adult mouse. This added feature of
reversibility provides greater control over the
timing and duration of a resulting phenotype,
and more closely mimics the effect of
pharmacological intervention. It also serves to
minimise the physiologic consequences of
specific gene modifications in the animal. GSK
has begun to employ these new technologies for
many of its mouse models.
Over 80% of mouse genes function the same as
those in humans. Mice also have a short
reproduction cycle and their embryos are
amenable to manipulation. Mice are therefore an
ideal human surrogate in the study of most
diseases. Currently over 95% of transgenic
animals used in biomedical research are mice.
Other transgenic animals include rats, pigs and
sheep. It is hoped that the refinement of
transgenesis techniques in mice will ultimately
allow for a corresponding reduction in the use of
"higher" animals, such as dogs and non-human
primates, in biomedical research.
3. GSK's use of transgenics
The total number of animals used by GSK has
been relatively stable over the last few years and
has recently seen a slight decline, despite huge
increases in research activity driven by
genomics and high throughput chemistry. The
decline is predominantly a result of GSK's
commitment to refinement and the development
of alternatives.
However, the revolution in genomics has meant
a rise in the proportion of animals used that are
transgenic, or involved in the transgenic
breeding process. The chart below illustrates the
fall in the total number of animals used relative
to 1995 and the increasing proportion of
transgenic animals in the UK.
Relative use of transgenics in GSK UK 1995 -
2003

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In the short term, the growing reliance on
transgenic animals in research may result in an
increase in the number of mice used. However,
possible changes to Home Office reporting
practices, to reflect a more accurate picture of
the total number of transgenics actually
developed and used for research purposes,
should serve to reduce numbers. Furthermore,
new emerging non-invasive imaging
technologies, such as ultrasound and magnetic
resonance imaging are being applied to evaluate
mouse models. These imaging tools allow
longitudinal measurements throughout the life-
time of animals and reduce the number of
animals required.
4. The human genome project and the role of
transgenics
Recent advances in molecular biology, and
particularly DNA sequencing, means that the
pharmaceutical industry can study most of the
30,000 genes - or potential drug targets - in the
human genome. This compares dramatically
with the 400 drug targets that the industry has
worked on over the past 50 years. Parallel
advances in high throughput in vitro screening
have helped to match this new genetic
knowledge more effectively with chemical
compounds. However, in vivo work remains a
fundamental element of the drug discovery and
development process.
5. The role of transgenic animals
Using transgenic animals gives rise to a number
of highly significant benefits.
Transgenic animals enable scientists to
understand the role of genes in specific diseases.
By either introducing or inactivating particular
genes, researchers can often for the first time
discover the root causes of diseases associated
with gene defects. For example, GSK scientists
engineered the overexpression of the human
mitochondrial transporter protein, "uncoupling
protein-3" (UCP-3), in skeletal muscle in mice.
In this model, the transgenic mice were found to
eat more than wild-type littermates, yet remain

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leaner and lighter. The mice also exhibit lower
glucose and insulin levels and an increased
glucose clearance rate, leading to the hypothesis
that compounds that regulate expression of
UCP-3 might be of use in treating obesity.
Transgenic animals allow more effective
treatments to be developed. Having found the
genes implicated in a disease, scientists can then
target these or design other therapies which act
by influencing their expression. For example,
studies in the 1990s suggested a role for the
enzyme cyclooxygenase-2 (Cox2) in the
inflammatory response. Targeted removal of the
Cox2 gene in mice prevented development of
autoimmune arthritis, thus validating the Cox2
enzyme as a good target for pharmaceutical
intervention.
Transgenic animals help test the safety of new
medicines and vaccines. Because transgenic
models can highlight specific characteristics
such as certain mechanisms involved in the
formation of tumours, they can demonstrate
more clearly the possible side effects of new
therapies. Their use in early toxicity trials may
also serve to prevent the subsequent use of a
larger number of animals in the development
phase.
Transgenics may spare the use of higher
animals. For example, GSK is currently awaiting
approval from the WHO for the use of a
transgenic mouse model (as an alternative to
non-human primates) for neurovirulence testing
of our Oral Polio Vaccine. Additionally,
transgenic mice have been successfully bred to
produce human CD4, a receptor found on the
surface of white blood cells. Since successfully
developing mice with this human receptor, GSK
has been able to eliminate the need to use
"higher" animals for testing drugs that interact
with the human CD4 receptor. This was
particularly important as chimpanzees, an
endangered and protected animal, are the only
other animals, other than humans, that carry the
CD4 receptor.
Transgenic animals can produce biological
products. It may be possible to use transgenic
animals to make rare biological products for
medical treatment. Human alpha-1-antitrypsin, a
protein used to treat the rare genetic disorder of
alpha-1-antitrypsin deficiency, is just one
example. Research is underway to breed

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transgenic sheep which produce the protein in
their milk. GSK is not involved in this area of
research.
Transgenic animals are being developed by
some companies to provide new organs for
transplantation such as kidneys, livers and
hearts. Transgenic pigs with human histo-
compatibility genes have been bred in the hope
that their "humanised" organs will not be
rejected by a patient's immune system. GSK is
not involved in this kind of research, which is
still very much in its infancy. If successful,
however, this research could transform the lives
of the many patients awaiting organ transplants.
6. Some perceptions around transgenic
animal experiments
Despite the importance of transgenic animals in
biomedical research, there are some concerns
and misconceptions raised about their use in
research. Some of these are addressed below:
Transgenic animals suffer more abnormalities
than regular research animals. The introduction
of DNA into an animal can be very complex and
the possible side effects can be difficult to
predict. Possible harms might arise from surgical
techniques used to harvest and re-implant
embryos; the collection of tissue from the tip of
the tail for genotyping; and non-specific effects
caused by damage to genes adjoining the altered
area of DNA. Also reduced fertility and/or
oversize foetuses may result from this
technology. In most cases the mutations impact
highly specific metabolic processes or cell
receptors without actually causing disease,
discomfort, pain or malformation in the animals.
The legal controls for their use are very stringent
and GSK devotes considerable resources to
monitoring these animals.
Transgenic animals not expressing foreign DNA
or not containing a particular gene modification
are destroyed. Because transgenesis is a complex
science, it is not 100% efficient. However, new
methods are being developed to increase the
accuracy in transgenesis. Again, it should be
remembered that such genetic alteration can
only be attempted if the authorities are
persuaded that there is no other way to pursue
important research.
The potential risks of transgenics to animals,
humans and the environment is too great to
justify their use. The UK has stringent

36
regulations which address these concerns. The
Genetic Modification of Organisms regulations
and the Environmental Protection Act (1990)
address the risks to those working with animals
and the impact on the environment of accidental
or planned releases. For example, no transgenic
animal is allowed to breed with wild populations
thus ensuring no long-term change in indigenous
populations.
The intrinsic worth of animals may be devalued
and their integrity violated by genetic
modification. Transgenic animals have not
chosen to have foreign DNA or other genetic
modifications. However, this potential "cost" to
the animals is routinely assessed under the
ethical review of proposed procedures and
weighed against the potential benefits. Medical
researchers only employ this technology when
no alternative research avenue exists. As the
Royal Society concluded in its 2001 Report
"The Use of Genetically Modified Animals", the
use of transgenic animals is fundamentally little
different from the use of other animals in
biomedical research. It is the degree of pain or
distress that is important, not the manner in
which the animal is bred. The presence of an
extra gene or a gene deletion does not
necessarily cause any suffering to the animal.
Conclusion
Gene-based biomedical research offers one of
the best hopes yet for curing the major diseases
which still afflict mankind. The use of transgenic
animals is central to realising that hope and
offers the potential for the use of fewer animals
in more targeted experiments. We must be clear.
There are only two alternatives to using animals.
One is to use humans in basic research; the other
is to delay or even give up the search for
desperately needed new treatments and cures.
The appropriate use of transgenic animals is a
positive development with potential for
significant medical benefits. The challenge is for
governments, industry and society to ensure that
transgenic research continues to be sensitively
carried out for proper medical ends in a suitably
balanced regulatory environment.

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