1

MINISTRY OF EDUCATION OF THE REPUBLIC OF AZERBAIJAN

BAKU STATE UNIVERSITY

Faculty: Biology
Department: Biochemistry and Biotechnology
4th course student on specialty
of Biology, group – 115 E
Quliyeva Lətafət Akif

COURSE WORK
on:
“Transgenic animals”

Defense date:
Evaluation:
Mark:

Scientific supervisor: MSc. N.Huseynova

Head of department: prof. Z.M.Mammadov

BAKU – 2020
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Kurs işinin tapşırıq vərəqi

Fakültə: Biologiya
Kafedra: Biokimya və Biotexnologiya
İxtisas: Biologiya
Bölmə: İngilis
Qrup:115 E
Tələbənin adı, soyadı, atasının adı: Quliyeva Lətafət Akif qızı
Mövzu: Transgenic animals
İş planı:

1. _________________________________________________________________
2. _________________________________________________________________
3. _________________________________________________________________
4. _________________________________________________________________
Tövsiyyə olunan ədəbiyyat:
1. “Transgenic Animal Science: Principles and Methods” (1991) Charles River
Laboratory. http://www.criver.com/techdocs/transgen.html
2. Hammer R.E, Pursel V.G, et al: Production of transgenic rabbits, sheep and pigs by
microinjection. Nature1985; 315(6021):680-683.
3. Jaenisch R: Germ line integration and Mendelian transmission of the exogenous
Moloney leukemia virus. Proc Natl Acad Sci.1976; 73:1260-1264.
4. Brackett B G, Boranska W, Sawicki W, Koprowski: Uptake of heterologous genome
by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl
Acad Sci.1971; 68:353-357.

Elmi rəhbər: N.Huseynova

Tapşırığın verilmə tarixi:
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CONTENTS
INTRODUCTION.....................................................................................................4
Chapter I. Transgenic animals as Biotechnology……………………………………5
Chapter II. Methods of creation of transgenic animals………………………………8
2.1. DNA microinjection……………………….……………………..……….......9
2.2. Embryonic stem cell-mediated gene transfer….…………………………….11
2.3. Retrovirus-mediated gene transfer…………………………………………...13
Chapter III. Application of transgenic animals…………………….………………14
CONCLUSION.........................................................................................................17
REFERENCES..........................................................................................................18
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INTRODUCTION
A transgenic animal is one whose genome has been changed by
the transfer from another species or breed of a gene or genes. Transgenic means that
one or more DNA sequences from another species have been introduced by artificial
means. Animals usually are made transgenic by having a small sequence of foreign
DNA injected into a fertilized egg or developing embryo. Transgenic plants can be
made by introducing foreign DNA into a variety of different tissues. Transgenic
animals are routinely used in the laboratory as models in biomedical research. Over 95
per cent of those used are genetically modified rodents, predominantly mice. They are
important tools for researching human disease, being used to understand gene function
in the context of disease susceptibility, progression and to determine responses to a
therapeutic intervention. Mice have also been genetically modified to naturally produce
human antibodies for use as therapeutics. Transgenic farm animals are also being
explored as a means to produce large quantities of complex human proteins for the
treatment of human disease. Such therapeutic proteins are currently produced in
mammalian cell-based reactors, but this production process is expensive.
A transgenic animal is where you take a piece of DNA that's not normally found in that
animal and place it back among its normal chromosomes. So for example, you can
make a transgenic by having a piece of DNA that you clone in a laboratory and inject
it into a fertilized egg of a mouse embryo, for example, then that becomes integrated
into the chromosome. And then when that mouse is born, it can transmit that extra piece
of DNA to its offspring. Transgenic animals can be used to model human diseases. So
for example, if there was a particular human disease that results from having a mutated
protein overexpressed, you can make a transgenic animal that also makes that same
mutated protein overexpressed. This then provides us with a mouse model that then
mimics that human disease that we can go into and actually study how that
overexpressed mutant protein causes the disease, and we can also use that animal to
test therapeutic interventions.
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Chapter I. TRANSGENIC ANIMALS AS BIOTECHNOLOGY

Transgenic animals are just one in a series of developments in the area of

biotechnology. Biotechnology has transformed the way in which we understand
processes such as engineering and manufacturing. These terms now include the use of
living organisms or their parts to make or modify products, to change the characteristics
of plants or animals, or to develop micro-organisms for specific uses. The novel uses
of biological techniques such as recombinant DNA techniques, cell fusion techniques,
mono and polyclonal antibody technology and biological processes for commercial
production have altered traditional distinctions and methods (US Congress, Office of
Technology Assessment, 1989). Genetic manipulations at the level of DNA have also
changed long held views as to what is considered to be animal, plant and human. In
turn, these changes have made it more difficult to evaluate the ways in which animals
are used and have obscured distinctions between pure and applied research.

Consideration of the acceptability of creating specific transgenic animal strains or

genetic manipulation involving interchanging DNA between species and kingdoms
could be a simple animal care issue or a societal decision. The following is an attempt
to show what the ability to create transgenic animals or engage in other forms of DNA
manipulation means in terms of traditional ACC functions, not forgetting that this
impacts on wider considerations of human responsibility for the welfare of other life
forms.

The creation of transgenic animals is resulting in a shift from the use of higher order
species to lower order species, and is also affecting the numbers of animals used. 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.
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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. 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, in addition to the numbers of animals which are sacrificed
as donors during the creation process. The potential of the technology has also made it
possible to consider employing cattle, swine, sheep and goats as processing units to
manufacture proteins or as organ donors.

The complex interactive processes of living mammals are not reproducible in vitro.
However, transgenic animals provide a means of evaluating genetic modifications in
terms of anatomical and physiological changes in a complex system. Transgenic
models are more precise in comparison to traditional animal models, for example the
oncomouse with its increased susceptibility to tumor development enables results for
carcinogenicity studies to be obtained within a shorter time-frame, thus reducing the
course of tumor development in experimentally affected animals. However, models are
not strict equivalents, so as with any other system care must be taken in drawing
conclusions from the data.

A representative, but non-inclusive, list of purposes for which transgenic animals have
been used indicates the wide ranging application of this biotechnology:

 in medical research, transgenic animals are used to identify the functions of

specific factors in complex homeostatic systems through over- or under-
expression of a modified gene (the inserted transgene);
 in toxicology: as responsive test animals (detection of toxicants);
 in mammalian developmental genetics;
 in molecular biology, the analysis of the regulation of gene expression makes
use of the evaluation of a specific genetic change at the level of the whole
animal;
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 in the pharmaceutical industry, targeted production of pharmaceutical proteins,

drug production and product efficacy testing;
 in biotechnology: as producers of specific proteins;
 genetically engineered hormones to increase milk yield, meat production;
genetic engineering of livestock and in aquaculture affecting modification of
animal physiology and/or anatomy; cloning procedures to reproduce specific
blood lines; and
 developing animals specially created for use in xenografting.

Important general considerations include the extent to which experience acquired in

the laboratory with regard to husbandry should influence industry standards for
keeping animals created specifically as living machines for the production of proteins,
antibodies, etc. What words are appropriate to describe and evaluate the condition of
animals now used as production units? The successful cloning of Dolly underlines the
fact that innovative developments in animal science are part of the mainstream of
biotechnology. In addition, the use of xenografts, at least at the public health level
makes animal and human welfare inseparable.
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Chapter II. METHODS OF CREATION OF TRANSGENIC ANIMALS

Transgenic animals are animals (most commonly mice) that have had a foreign
gene deliberately inserted into their genome (Fig.1). Such animals are most
commonly created by the microinjection of DNA into the pronuclei of a fertilised egg
which is subsequently implanted into the oviduct of a pseudopregnant surrogate
mother. For practical reasons, i.e., their small size and low cost of housing in
comparison to that for larger vertebrates, their short generation time, and their fairly
well defined genetics, mice have become the main species used in the field of
transgenics. The three principal methods used for the creation of transgenic animals
are DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirus-
mediated gene transfer.

Figure 1. The photo shows two transgenic mice positioned either side of a plain mouse.
The transgenic mice have been genetically modified so that they carry a green
fluorescent protein which glows green under blue light.
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2.1. DNA MICROINJECTION

Microinjection is a useful technique that plays an important part in the generation

of genetically modified mouse and rat models. Different microinjection systems can
exist, the construction and configuration of which directly depends on the type of
experiment. At Creative Biolabs, we are now offering DNA microinjection for
transgenic model creation, ES cell injection and CRISPR injection services for
customers with a need. 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. 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.

A major advantage of this method is its applicability to a wide variety of species.

DNA microinjection (Fig.2) is the dominating technique leading to random integration

of a transgene via the introduction of DNA into the pronucleus of a developing zygote.
Following fertilization of a mouse egg, the male and female pronuclei remain separated
for a few hours before they fuse to make the zygotic nucleus, thus allowing for
microinjection of the desired genes into the larger male pronucleus. Eggs that can
survive the injections are transferred into the oviducts of a foster mother i.e.,
pseudopregnant female mice for the generation of Founder mouse, from which
permanent transgenic lines can be established. The presence of transgenes in the
offsprings can be identified by PCR analysis or Southern blot hybridization.
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Two kinds of DNA injections are included in our list:

 Plasmid Vectors
 BAC Injections
Features of DNA Microinjection:

 The amount of DNA delivered per cell is not limited and the delivery is precise, both
of which increase the chance for integrative transformation.
 As a direct physical approach, this technique is host-range independent.
 Cells that are injected with the DNA material have high regeneration potential.
 Although pronuclear microinjection is popular, this method for transgenesis has
some disadvantages such as low success rate and mosaic founders.

Figure2. Diagram of DNA Microinjection into a Pronucleus.

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2.2. EMBRYONIC STEM CELL-MEDIATED GENE TRANSFER

Embryonic stem cell-mediated gene transfer (Fig.3) is the introduction of DNA
into embryonic stem cells. (ES cells). Embryonic stem cells can differentiate into all
types of cells when introduced to another embryo. DNA introduced into embryonic
stem cells may integrate randomly, just like in pronuclear micro-injection. If the
introduced DNA is similar in sequence to part of the animal genome, it may undergo
“homologous recombination” and integrate as a single copy at a specific site. ES cells
will colonize a host embryo and often contribute to the germ line. This results in the
production of some sperm carrying the extra DNA. When these transgenic sperms
fertilize a normal egg, a transgenic animal will be produced with the same foreign DNA
in every cell.

The method involves:

 Using recombinant DNA methods, build molecules of DNA containing the structural
gene you desire (e.g, the insulin gene), vector DNA to enable the molecules to be
inserted into host DNA molecules, promoter and enhancer sequences to enable the gene
to be expressed by host cells.
 Transform ES cells in culture to expose cultured cells to the DNA so that some will
incorporate it.
 Select for successfully transformed cells.
 Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
 Embryo transfer.
 Prepare a pseudopregnant The stimulus of mating elicits the hormonal changes needed
to make her uterus receptive.
 Transfer the embryos into her uterus.
 Hope that they implant successfully and develop into healthy pups (no more than one-
third will).
 Test her offspring.
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 Remove a small piece of tissue from the tail and examine its DNA for the desired gene.
No more than 10- 20% will have it, and they will be heterozygous for the gene.
 Establish a transgenic strain
 Mate two heterozygous mice and screen their offspring for the 1:4 that will be
homozygous for the transgene.
 Mating these will found the transgenic strain.

Figure 3. Embryonic stem cell-mediated gene transfer.

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2.3. RETROVIRUS-MEDIATED GENE TRANSFER

In transgenic animal production, gene transfer efficiency is the limiting factor
in transgenezis success rates. Among many gene transfer systems developed to date,
the retrovirus vector-mediated gene transfer system has been an unequalled choice in
gene transfer efficiency. The most important features of retroviruses in regard to their
use as vectors are the technical ease and effectiveness of gene transfer, due to their
affinity and infectivity for certain target cells, leading to
successful transgene incorporation. Once cells are infected by retroviruses, the
resultant viral DNA, after reverse transcription and integration, becomes a part of the
host cell genome and is maintained for the life of the host cell. In addition, it is believed
that DNase hypersensitive regions are the preferred targets for retrovirus integration
implying efficient expression of exogenous proviral genes even though the proviral
copy number for each integration site is limited to a single copy. Unlike
DNA microinjection, integration of a viral gene does not seem to induce
rearrangements of the host genome. In this chapter, basic principles of the retrovirus
system are discussed, as are current problems to be resolved for application to
transgenic animal production.
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Chapter III. APPLICATION OF TRANSGENIC ANIMALS

As disease model: Historically, mice have been used to model human disease
because of their physiological, anatomical and genomic similarities to humans.
Transgenic animals are produced as disease models (animals genetically manipulated
to exhibit disease symptoms so that effective treatment can be studied) such as
Alzheimer, cancer, AIDS. Transgenic animals enable scientists to understand the role
of genes in specific diseases. The benefits of using transgenic animals include the
possibility of the replacement of higher species by lower species- through development
of disease models in mice rather than in dogs or non-human primates, the extent of
discomfort experienced by parent animals during the experimental procedures.
Transgenic animals such as mice have been found to be valuable in investifations into
gene function and for analysis of different hereditary diseases.
As food: The FDA suggested that cloned animals and their products were safe to eat
for human being. Some drawbacks are associated due to their muscle hypertrophy like
difficulties in calving requiring Caesareans, poor viability of calves and poor fertility.
Drug and Industrial production: Transgenic animals are used for production of proteins
such as alpha-1-antitrypsin, produced in liver, used in treatment of emphysema or
cystic fibrosis. This process is less expensive than production of protein through culture
of human cells. The human lungs are constantly get affected by foreign particles such
as dust, spores and bacteria. To prevent these, neutrophils releasing the elastase enzyme
but this enzyme harmed the elastin in the lungs which maintains the elasticity of lungs.
So, human body releases a protein α1 proteinase inhibitor which has been successfully
expressed in sheep. Recombinant human proteins produced in the mammary glands of
transgenic animals. Pharmaceutical proteins are now used for commercial purpose.
Two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells
of lactating goats. The goats are used to manufacture silk, milk and secrete tiny strands
from their body by the bucketful. By extracting polymer strands from the milk and
weaving them into thread which is light and tough material that could be used to
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prepare military uniforms, medical micro sutures and tennis racket strings. Americans
are more supportive (60%) for above use of transgenic animals. The mammary gland
of transgenic goats is used to produce Monoclonal Antibodies. A recombinant
bispecific antibody is produced by using transgenic cattles with in their blood 48.
Another application includes newly generation of trans-chromosomal animals in which
a human artificial chromosome containing the complete sequences of the human
immunoglobulin heavy and light chain loci was introduced into bovine fibroblasts,
which were then used in nuclear transfer. Transchromosomal bovine offspring were
obtained that expressed human immunoglobulin in their blood. This could be a
significant step forward in the generation of human therapeutic polyclonal antibodies.
Disease control: Scientist developed the mice by altering the genes of the mousepox
virus in Australia. Some scientist also thought to develop genetically modify
mosquitoes so they cannot produce malaria but other scientist worry about these
mosquitoes that they could have unforeseen possibly risk if, they are released into the
environment.
Xenotransplantation: Now a day approximately about 250000 people are alive due to
the successful transplantation of an appropriate allotransplantation. Sometimes there is
limitation of appropriate organs or rejection of live organ donation. So, to rectify this
problem porcine xenografts from domesticated pigs are considered to be the best
choice. Pigs which are genetically modified can be used as a source animal for tissues
and organs in human beings for transplantation purpose by delete the gene responsible
for the human rapid immune rejection response. In Canada, a National survey on
xenotransplantation showed that only 48% found acceptable for ‘the use of animals as
a source of living cells, tissues or organs to prolong human life. To overcome the
Hyperacute rejection & acute vascular rejection, synthesis of human regulators of
complement activity is produced in transgenic pigs. Survival rates, after the
transplantation of porcine hearts or kidneys expressing transgenic regulators of
complement activity proteins to immunosuppressed nonhuman primates, reached near
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about 23 to 135 days. So, the Hyperacute rejection can be overcome in a clinically
acceptable manner. For long term graft tolerance induction of permanent chimerism
via intraportal injection of embryonic stem (ES) cells or the co-transplantation of
vascularised thymic tissue.
Blood replacement Transgenic swine are used to produce human haemoglobin. The
protein obtained from transgenesis could be purified by using procine blood which is
similar to human haemoglobin.
Agriculture Transgenic pigs containing a human metallothionein promoter or porcine
growth-hormone gene construct referred significant improvements in economically
traits including growth rate, body fat/muscle ratio. Transgenic pigs are used to produce
pork by using spinach desaturase gene which produce large amount of non-saturated
fatty acids, used for diet purpose and was advantageous to reduce the risk of stroke and
coronary disease. Transgenic animals are used for milk production. Generally, there is
an improvement in milk composition. For this purpose transgenic mice have been
developed, at the same time some unwanted side effects can occur. Transgenic pigs are
used to increase milk production by altering the composition of lactose. In the pig,
transgenic expression of a bovine lactalbumin construct in sow milk has been resulting
in higher lactose contents and greater milk yields, correlated with improved survival
and development of piglets. Transgenic sheep are used for wool production in which
transgenic sheep carrying a keratin-IGF-I construct showed that expression in the skin
and the amount of clear fleece was about 6.2% greater in transgenic as compared to
nontransgenic animals. 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.
Transgenic animals are used in toxicity testing. Transgenic animals are used for
vaccine testing.
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CONCLUSION
Transgenic animal has made significant contributions to human health and well-
being. The recent advances in reproductive technologies (in vitro production of
embryos, sperm sexing, somatic nuclear transfer, Lentiviral transfer of oocytes and
zygotes, Chimera generation by injecting the pluripotent cells) adds a new dimension
to animal breeding. The application of transgenic animals showed that within the next
five to eight years genetically modified animals will play a significant and important
role in the biomedical field, in particular via the production of valuable
pharmaceutical proteins and the supply of xenografts. New and exciting techniques
being developed will continue to expand this important and useful area of
experimentation.
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REFERENCES
1. “Transgenic Animal Science: Principles and Methods” (1991) Charles
River Laboratory. http://www.criver.com/techdocs/transgen.html
2. Hammer R.E, Pursel V.G, et al: Production of transgenic rabbits, sheep
and pigs by microinjection. Nature1985; 315(6021):680-683.
3. Jaenisch R: Germ line integration and Mendelian transmission of the
exogenous Moloney leukemia virus. Proc Natl Acad Sci.1976; 73:1260-
1264.
4. Brackett B G, Boranska W, Sawicki W, Koprowski: Uptake of
heterologous genome by mammalian spermatozoa and its transfer to ova
through fertilization. Proc Natl Acad Sci.1971; 68:353-357.