Paper No.

: 16 Molecular Genetics
Module : 17 Transgenic Animals: Applications

Development Team
Principal Investigator : Prof. Neeta Sehgal
Department of Zoology, University of Delhi

Co-Principal Investigator : Prof. D.K. Singh

Department of Zoology, University of Delhi

Paper Coordinator : Prof. Namita Agarwal

Department of Zoology, University of Delhi

Content Writer : Dr. Kamal Kumar Gupta,

Deshbandhu College, University of Delhi

Content Reviewer : Dr. Surajit Sarkar

Department of Genetics, South Campus, University of Delhi

ZOOLOGY Molecular Genetics

Transgenic animals: Applications
 Description of Module

Subject Name ZOOLOGY

Paper Name Zool 016: Molecular Genetics

Module Name/Title Genetically Modified Organisms

Module ID M17: Transgenic Animals: Applications

Keywords

Contents
1. Learning Outcomes
2. Introduction
3. Transgenic Animals: Models for Determining Biological Basis of Diseases
3.1. Transgenic Animal Models for Genetic Diseases
3.1.1. Transgenic Mouse Models for Alzheimer Disease
3.1.2. Transgenic Mouse Model for Huntington Disease
3.2. Transgenic Mouse Models for Infectious Diseases
3.3. Transgenic Mouse Model to Study Effects of Cell Death
4. Medical Applications of Transgenic Animals
4.1. Production of Pharmaceuticals
4.2. XenoMouse: Production of Fully-Human Monoclonal Antibodies
4.3. Production of Donor Organs: Xenotransplantation
5. Improving Nutritional Quality
5.1. Improving Milk Quality of Dairy Cattle
5.2. Enhancement of omega-3 fatty acid in Pig
6. Environment Friendly Transgenic animals
6.1. Enviropig: Environment Friendly Pig
6.2. Medaka: Pollution Monitoring
7. Disease Resistant Transgenic Livestock
8. Transgenic Poultry
9. Transgenic Fish
10. Summary

ZOOLOGY Molecular Genetics

Transgenic animals: Applications
 1. Learning Outcomes
After studying this module, you will learn that transgenic animals have been widely used in
research for understanding biological basis of genetic and infectious diseases. Many
transgenic animals such as Glo Fish, Aqu Advantage-salmon and other have been created and
approved for human use. Knockout Mouse Project (KOMP) aimed to produce mouse with
knockout mutation in each of the over 20,000 genes in the mouse genome. Transgenic
livestock has been used and approved for production of pharmaceuticals and therapeutics. In
future Transgenesis can be used in production of disease resistant livestock, environmental
friendly organisms, organs for xenotransplantation, monoclonal antibodies etc.

2. Introduction
Transgenesis has wider applications in improving genetic features of domesticated animals
and production of human pharmaceutical in the farm animals. Transgenic animals are suitable
models for study of human diseases. Study the genes regulation, tumor development,
immunological specificity molecular genetics of development of animals can also be studied
on transgenic animals.

Oversize mice containing a human growth hormone transgene was one of the first transgenic
animals created (Fig. 1). “Astrid,” the first transgenic was pig created in 1992. This opened
new vistas for xenotransplantation of organ to human. The first genetically modified animal
to be commercialized was the GloFish, a Zebra fish with a fluorescent gene allows it to glow
in the dark under ultraviolet light. Aqu Advantage salmon was the first genetically modified
animal approved for food use.

Fig. 1: Mice from Dr. Ralph L. Brinster's famous giant mouse experiment, in which the rat growth hormone
gene was expressed in the liver of mice

ZOOLOGY Molecular Genetics

Transgenic animals: Applications
 Transgenic farm animals can be used as bioreactors to produce useful pharmaceutical
products. Some of the major thrust areas in application of transgenic animals include
increasing nutritional value of milk, protecting farm animals against common pathogens that
cause disease and animal loss.

3. Transgenic Animals: Models for Determining Biological Basis of

Diseases
The transgenic animal models help to understand the molecular basis of disease, and reveal
some potential targets for their treatment.

3.1. Transgenic Animal Models for Genetic Diseases

Transgenic mouse models have been developed for human genetic diseases, such as
Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, arthritis, muscular
dystrophy, tumorigenesis, hypertension, neurodegenerative disorders, endocrine dysfunction,
and coronary disease etc. Knockout Mouse Project (KOMP) was initiated in 2006 with the
goal of producing knockout mutation in the genes of the mouse genome. At present it is
coordinated by the International Mouse Phenotyping Consortium (IMPC). The knockout
mice provide critical tools for understanding gene function and the genetic causes of human
diseases (Fig. 2).

Fig. 2: A laboratory mouse in which a gene affecting hair growth has been knocked out (left)
Source: https://en.wikipedia.org/wiki/Knockout_mouse

3.1.1. Transgenic Mouse Models for Alzheimer Disease

Alzheimer disease is a chronic neurodegenerative disorder that is characterized by dementia,

the progressive loss of abstract thinking, memory and intellectual abilities. It results in

ZOOLOGY Molecular Genetics

Transgenic animals: Applications
 personality change, language disturbances, and a slowing of physical capabilities which
interfere with daily life

Fig. 3: Pathology of Alzheimer disease neuron

Source: https://in.pinterest.com/joanbeuerlein/alzheimers-dementia/?lp=true

The patients show accumulation of neurofibrillary tangles within the cell bodies of the
neurons, development of dense extracellular aggregates called senile plaques at the ends of
inflamed nerves, and loss of neurons in the neocortex and hippocampus of the brain (Fig. 3).
The core of a senile plaque is composed of a fibrillar structure called amyloid body. The
amyloid bodies contain Aβ proteins which are derived from an internal proteolytic cleavage
of the β-amyloid precursor protein (APP). Faulty cleavage of the APP protein causes the
production of Aβ40 and Aβ42, the main variants in Alzheimer disease. Inefficient clearance
of the variants likely leads to their accumulation (Fig. 4).

Fig. 4: Formation of senile Amyloid plaques in Alzheimer disease (Source: Author)

Mouse models for Alzheimer disease were created with transgenes that contain mutations in
the APP gene. Two mutant genes of APP, APP-717 and APP-670/671 were used creation of
transgenic mouse model for Alzheimer disease. APP-717 contains phenylalanine instead of
valine and APP-670/671 contains asparagine and leucine instead of lysine and methionine. A
transgene with the APP-717 mutation was constructed from an APP cDNA. Modified introns

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Transgenic animals: Applications
 were added between exons 6 and 7, 7 and 8, and 8 and 9 of the APP cDNA. Presence of
intron in transgene increases the rate of transcription. The promotor sequences from platelet-
derived growth factor which express in brain were taken for “APP cDNA–intron” construct.
This complete construct is called the PDAPP minigene (Fig. 5).

Fig. 5: Structure of PDAPP minigene (Source: Author)

The transgenes were introduced in the mice. The transgenic mice containing about 40 copies
of the PDAPP minigene on ageing display amyloid plaques, neuronal cell death, and memory
defects. Mice with APP-670/671 gene construct also produces Alzheimer disease-like
features. The formation of amyloid plaques in humans has also been shown to be associated
with increased production of a protein BACE1 (β-site APP cleaving enzyme 1) protease that
cleaves APP to produce Aβ. Transgenic mice that carry a knockout mutation in BACE1
produce Aβ but do not develop Aβ amyloid plaques. BACE1 knockout mice exhibit
deleterious behavioral defects, which indicate that some BACE1 is required for normal
development and/or normal adult brain activity. Reduced production of BACE1 by RNAi
therefore, may prove a treatment to reduce or delay Alzheimer disease. shRNAs that target
BACE1 mRNA were carried on a lentiviral vector that was injected into the hippocampus in
transgenic mice. These mice showed a reduction in the Aβ deposits and plaque formation.

3.1.2. Transgenic Mouse Model for Huntington Disease

Huntington disease is an incurable, fatal neurological genetic disorder. It remains confined to

specific regions of the brain. About 1 in 10,000 people worldwide are affected by this
disease. The gene is due to change in HD gene, codes the huntingtin protein. It has been seen
that addition of CAG trinucleotides units to exon 1 of the HD gene is responsible for the
disease. During translation CAG code for glutamine, consequently, multiple CAG codons
incorporate a series of glutamine residues (polyglutamine) in the huntingtin protein.
Symptoms of Huntington disease occur when the number of CAG codons in polyglutamine
region is 38 or more.

ZOOLOGY Molecular Genetics

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 Fig. 6: transgenic mouse model of Huntington disease carrying a mutant form of the HD gene. Huntingtin
protein is expressed under the control of the tet-off system. CAG94, a sequence of 94 CAG repeats. pFB,
forebrain-specific promoter; tTA, tetracycline transactivator; tetO, tetracycline operator; p, promoter.
Source: Author

A mouse model for Huntington disease was created using „tet-off‟ conditional regulation
system (Fig. 6). HD genes that contain exon 1 with 94 CAG repeats as the transgene. The
tTA gene was placed under the control of a promoter that is active in the cells of the
forebrain. Expression of HD transgene was switched off in the embryos during pregnancy by
adding doxycycline to the drinking water. After birth, doxycycline was not supplied to the
transgenic mice. This allowed continuous expression of the mutant HD gene and the
production of a protein with a long polyglutamine sequence. A neurological condition that
was similar to Huntington disease in humans was developed in these transgenic mice. The
features of the disease disappeared when the expression of the mutant HD gene was
prevented by the addition of doxycycline. This indicates that a continuous expression of a
mutant HD gene is required for establishment of the disease. The brain cells can recover
when this synthesis of HD protein ceases.

Transgenic primate models, such as the rhesus macaque, have also been developed for such
human neurodegenerative diseases for better understanding.

3.2. Transgenic Mouse Models for Infectious Diseases

Pseudorabies virus is an alpha herpes virus that infects pigs. Viral infection result in
encephalitis and respiratory illness in young pigs and abortion and infertility in sows.

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Transgenic animals: Applications
 Nectin-1 is a porcine receptor to which alpha herpes virus bind. Entry of the virus into host
cells can be blocked by expressing a soluble form of this host cell receptor i.e. Nectin-1. This
would prevent the virus from binding to the host membrane-bound receptor; hence prevent
viral penetration of the host cell.

Before the creation of transgenic farm animals, it was tested for its ability to protect against
pseudorabies infection in a mouse model. A transgene was constructed from the DNA
sequence encoding the extracellular domain of the nectin-1. This was fused to the gene for
the constant region of human immunoglobulin G (IgG). It was placed under the control of a
promoter so that it can express in several cell types. This fusion construct could produce a
secreted form of the nectin-1. The transgenic mice were found resistance pseudorabies virus.
Moreover, antibodies against the virus were not detected in the transgenic mice. These
studies demonstrate that expression of a secreted form of the pseudorabies receptor in
transgenic mice can protect the animals against viral infection. In this way transgenic pigs
can be produced that resist pseudorabies virus infection.

3.3. Transgenic Mouse Model to Study Effects of Cell Death

In human beings the diphtheria toxin is produced by the bacterial pathogen Corynebacterium
diphtheria. It binds to the heparin-binding epidermal growth factor receptor present on the
cell. This toxin–receptor complex is then taken up into the cell, where it inactivates
elongation factor 2 (EF-2) required for protein synthesis. Failure of protein synthesis leads to
cell death (Fig. 7).

Fig. 7: Genetically engineered cell death. (A) Human heparin-binding epidermal growth factor receptor (HB-
EGFr) is synthesized in liver cells from an HB-EGFr transgene under the control of a liver cell-specific
promoter (pliver). (B) Diphtheria toxin binds to HB-EGFr and is taken into the cell. This inactivates EF-2 and
cause cell death.
Source: Author

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Transgenic animals: Applications
 The cell death can be induced in transgenic mice in order to study organ failure resulting
from cell death. Mouse cells are not normally susceptible to diphtheria toxin because they do
not have a receptor that recognizes the bacterial protein. Transgenic mice were engineered to
express the human heparin-binding epidermal growth factor receptor under the control of a
liver-specific promoter. This results in expression of HBEGF – receptors in the cell
membrane of the liver cells (Fig. 7). Treatment of these transgenic mice with diphtheria toxin
leads to liver damage. The presence of human heparin-binding epidermal growth factor
receptor in the cell membrane of the mouse liver cells had no effects on liver cell functions or
other processes in the absence of the diphtheria toxin.

4. Medical Applications of Transgenic Animals

4.1. Production of Pharmaceuticals

The mammary glands of the dairy cattle can be used as a bioreactor for the production of
pharmaceutical proteins and therapeutic agents. Many transgene constructs that have
mammary gland-specific promoters and human gene sequences has been successfully
introduced and expressed in the milk of transgenic sheep, goats, pigs, and rabbits. The
advantage of the transgene-derived proteins is that these proteins are glycosylated and have
other posttranslational modifications. The proteins secreted in the milk usually have
biological activities similar to human proteins.

Strategy for expressing human genes in the milk of domestic animal

To express the human hormone gene in cattle milk, the coding sequence of the human
hormone gene is linked with the promoter of β-lactoglobulin gene, a gene is normally
expressed in mammary glands cells. In addition a short signal sequence, necessary for protein
secretion in the milk was also included in the transgene. These transgenes are incorporated in
the sheep. In this way transgenic sheep producing human hormone in their milk can be
created. The hormone can be purified from the milk and used to treat humans.

Transgenic cattle have wide potential in production of pharmaceuticals and therapeutics.

High yielding varieties of cattle can produce approximately 10,000 liters of milk annually. If
amount of recombinant protein in the milk is 1 gram per liter of milk and it could be purified
with 50% efficiency, 20 transgenic cows would yield about 100 kg of the recombinant
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 protein per annum. This much yield suffices the annual global requirement for protein C,
which is used for the prevention of blood clots. Similarly one transgenic cow would be
sufficient for the production of the annual world supply of factor IX (plasma thromboplastin
component), which is used by hemophiliacs to facilitate blood clotting.

Transgenic goats and sheep can also be raised to produce pharmaceuticals in their milk.
Recently U.S. Food and Drug Administration approved the human protein „antithrombin‟
produced in transgenic goat‟s milk for the use in the individuals with a hereditary deficiency
for this protein. Antithrombin is an anticlotting factor, prevents the excessive formation of
blood clots, by inhibiting the activity of thrombin. Approximately 1 in 5,000 people is unable
to produce this protein naturally. Therefore, they are at risk for heart attacks and strokes.
Conventionally the antithrombin is extracted from the plasma of donated blood. This has
higher risk of contamination with pathogens. Also process of extraction is less efficient and
more costly; the supply is also not sufficient to meet the needs of patients. The milk of
transgenic goats is a significant source of human antithrombin, which yields 2 to 10 grams
per liter of milk. It has been estimated that 75 transgenic goats are sufficient to meet the
annual worldwide demand for antithrombin. Many other human therapeutic proteins such as
antitrypsin, human clotting factors (factor IX for the treatment of hemophilia) and
monoclonal antibodies have also been expressed in transgenic goats.

4.2. XenoMouse: Production of Fully-Human Monoclonal Antibodies

In theory, monoclonal antibodies can be effective agents for diminishing the proliferation of
cancer cells and treating other human diseases. However, it is impossible to generate human
monoclonal antibodies. The rodent monoclonal antibodies are immunogenic to humans and
elicit anti-mouse antibodies that result in destruction of the therapeutic antibody.
Recombinant DNA strategies have been devised to “humanize” existing rodent monoclonal
antibodies.

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 Fig. 8: Creation of Xenomouse
Source: https://in.pinterest.com/pin/325736985531240468/

An antibody is a tetrameric protein with one pair of the heavy chain and one pair of light
chain. The genetic information for a specific heavy chain is created by rearrangement of
several heavy-chain-specific DNA segments in a B cell. Two light chains are encoded by
DNA rearrangements of other, light-chain-specific DNA segments. Each single B cell
synthesizes only one kind of antibody molecule that has a unique set of rearranged segments
for a heavy chain and a light chain. The genetic repertoire for the formation of the vast
numbers of different human antibodies consists of more than 100 heavy-chain DNA segments
and a similar number of light-chain DNA segments. To create a transgenic mouse that is
capable of synthesizing a full range of human antibodies against every antigen, the
endogenous mouse heavy and light chain genes were inactivated, and YACs carrying most of
the heavy and light-chain DNA elements from each human immunoglobulin gene were
inserted into the chromosomal DNA of the mouse (Fig. 8). A commercialized version of the
human antibody producing mouse has been designated the XenoMouse. First fully human
monoclonal antibody produced in this mouse (Panitumumab) has received regulatory
approval for use as a treatment for advanced colorectal cancer. Other therapeutic antibodies
produced in the XenoMouse, including several for the treatment of various cancers and
osteoporosis, are now in clinical trials.

4.3. Production of Donor Organs: Xenotransplantation

Transgenic animals can be used as a potential source of organs for transplantation into human
beings. Organ transplant is recommended in case of organ failure. Currently, organs such as

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 hearts, livers, and kidneys are taken from donor and transplanted in the recipient. However,
demand for donated organs far exceeds the available supply. Animal-to-human transplants
(xenotransplantation) can be a way to supply organ for transplantation. Pig organs can be
considered for xenotransplantation as they are similar in size and physiological functions to
those of humans.

A major limitation of xenotransplantation is hyperacute rejection of the animal organ by the

recipient. It is due to the binding of preexisting antibodies of the recipient to a carbohydrate
epitope (α-Gal) present on the surfaces of the cells of the transplanted organ. This elicits an
inflammatory response that destroys the transplanted organ.

It was proposed if the donor animal carried one or more of the genes for a human
complement-inhibiting proteins, a transplanted organ would be protected from the initial
inflammatory response. Transgenic pigs with different human complement inhibitor genes
have been produced. Hyperacute rejection did not occur in the primate recipient after kidneys
from transgenic pigs were transplanted; survival period was 20 to 90 day.

Another strategy for xenotransplantation is to produce transgenic pigs with the organs that do
not produce the antigenic α-Gal epitope by deleting the gene encoding 1, 3-α-galactosyl
transferase.

5. Improving Nutritional Quality

5.1. Improving Milk Quality of Dairy Cattle

Transgenic dairy cattle with improved nutritional value of milk for humans and for suckling
can be produced. Overexpression of proteins in the milk can improve the growth, health, and
survival of suckling animals.

Specific components of milk can also be altered as per human requirement. Cheese
production from milk is directly proportional to the β-casein and κ-casein contents. The
amount of these proteins can be increased in milk of the cows engineered with additional
copies of the β-casein and κ-casein genes. Transgenic cows can also be created having a
higher concentration of some amino acids and a lower fat content in the milk. This increased
its nutritional value.

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 Lactose-intolerant individuals lack lactose-hydrolyzing enzyme lactase and therefore,
experience severe indigestion after the consumption of milk. Decrease in lactose content of
milk can be achieved by expression of the mammalian lactase gene in the mammary glands.

Many people are allergic to β-lactoglobulin present in bovine milk. Knock out mutant of β-
lactoglobulin can be created to remove β-lactoglobulin in the milk.

5.2. Enhancement of omega-3 fatty acid in Pig

Omega-3 fatty acids are long-chain polyunsaturated fatty acids found mainly in fishes.
Humans and other livestock animals cannot produce these fatty acids. The livestock animals
contain high levels of omega-6 fatty acids. They cannot convert omega-6 fatty acids to
omega-3 fatty acids as they lack the enzymes desaturase. Diets with high omega-6 content
can cause many diseases such as cancer, heart disease, and diabetes.

Transgenic pigs that synthesize omega-3 fatty acids can be produced. The roundworm
Caenorhabditis elegans produces an enzyme desaturase that converts omega-6 fatty acids to
omega-3 fatty acids by introducing a double bond into the hydrocarbon chain. A transgene
was created containing enzyme desaturase gene (fat-1) from C. elegans. The gene was cloned
into an expression vector under the control of the chicken β-actin promoter and the
cytomegalovirus enhancer. Foetal pig fibroblasts were transfected and cultured. The cultured
cells that produced higher levels of omega-3 fatty acids were used to produce fat-1 transgenic
pigs by nuclear transfer. The transgenic pigs showed threefold-higher levels of omega-3 fatty
acids and 23% lower levels of omega-6 fatty acids than nontransgenic pigs.

6. Environment Friendly Transgenic animals

6.1. Enviropig: Environment Friendly Pig

Enviropig is the trademark for a genetically modified line of Yorkshire pigs, with the
capability to digest plant phosphorus more efficiently than conventional unmodified pigs.
These transgenic pigs were developed at the University of Guelph.

The main food source for pigs is soybean meal, which has about 50% or more of its
phosphate in the form of phytate. The pigs are unable to digest and utilize the phytate due to
the absence of the enzyme phytase. Therefore, they excrete large amounts of phosphorus

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 which causes serious environmental problem. The phosphorus from the pig manure can run
off into water systems and cause excessive growth of cyanobacterial and algal populations.
This result in depletion of the oxygen supply and subsequently kill fish and other aquatic
organisms. Presence of large amounts of phosphorus in the environment also causes
production of greenhouse gases that contribute to global warming.

Fig. 9: Enviropig with a transgene consisting of the phytase gene appA from E. coli and the parotid secretory
protein promoter (Source: http://mmg-233-2014-genetics genomics.wikia.com/wiki/EnviroPigs)

The enzyme phytase is found in plants and microorganisms which removes phosphates from
phytate. A transgene consisting of the phytase gene appA from E. coli and the parotid
secretory protein promoter was constructed (Fig.). Transgenic pigs were created by
pronuclear microinjection. The transgenic pigs produce the enzyme phytase in the salivary
glands that is secreted in the saliva. The phytase mixes with the feed and become active in the
acidic environment of the stomach and digest phytate present in the feed (Fig.).
Consequently, there is less phosphorus in the manure; hence it is environment friendly.

6.2. Medaka: Pollution Monitoring

Synthetic derivatives of natural estrogens are used in most oral contraceptives, as a therapy
for postmenopausal disorders in women, to treat infertility and endometriosis, and to develop
female-only fish populations in aquaculture. A wide variety of industrial chemicals, such as
bisphenol A and polychlorinated biphenyls (PCBs), also have estrogenic activity in animals.
A large amount of these estrogenic chemicals are flushed into aquatic ecosystems with
domestic, agricultural, and industrial wastewater and cause water pollution.

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 Fig. 10: Transgenic medaka with green fluorescent protein (gfp) under estrogen responsive promoter from
vitellogenin gene (pvit)
Source: Author

Transgenic medaka has been developed to monitor quality of water and detect estrogenic
compounds in aquatic environments. A transgene was constructed for green fluorescent
protein under the control of estrogen responsive promoter from the medaka vitellogenin gene
(Fig.) The gene was cloned and injected into medaka eggs. Vitellogenin is normally
synthesized in females in response to endogenous estrogens. Exposure of transgenic fish to
17β-estradiol and other natural and synthetic estrogenic compounds activates the vitellogenin
promoter. This leads to production of green fluorescent protein that can be visualized as
emission of green fluorescence in the living fish (Fig. 10).

A variation of this transgenic model can be used for the assessment of heavy metal
contamination of water. A heavy-metal-inducible promoter can be incorporated adjacent to
the red fluorescent protein gene. This transgene can be used to create transgenic zebra fish.
When these transgenic zebrafish are kept in water contaminated by mercury and other heavy
metals, the promoter becomes activated, inducing expression of the red fluorescent protein
gene.

7. Disease Resistant Transgenic Livestock

Transgenic animals which are resistance to infectious diseases have been developed. Mastitis
(mammary gland abscesses) in dairy cattle, bovine spongiform encephalopathy (BSE) also
known as mad cow disease in cattle and neonatal scours (dysentery) in swine are some of the
target diseases for production of transgenic livestock.

In Vivo Immunization: Transgenes encoding the heavy and light chains of a monoclonal
antibody have been introduced into recipient animals. This concept is called in vivo
immunization. Expression of a monoclonal antibody against a specific pathogen provides
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 immediate protection without prior exposure to the pathogen. If the monoclonal antibody is
secreted into milk, young suckling animals also acquire passive immunity against a pathogen.
Another approach to produce disease resistant livestock is elimination of the host cell
component to which the infectious agent interacts

Bovine Spongiform Encephalopathy (BSE): Bovine Spongiform Encephalopathy (BSE)

also known as mad cow disease is a neuropathological disorder in cows. The brain tissue of
infected animals becomes filled with holes that give the brain a characteristic sponge-like
appearance. It is caused by a mutant form of the prion protein. The mutant prion proteins
induce the brain proteins to misfold. The misfolded proteins aggregate and disrupt normal
brain function. There is no known treatment for the disease, and therefore, the infected
animals have to be destroyed. Moreover, the prions can be transmitted to humans through
consumption of prion-contaminated meat and can cause a variant form of encephalopathy.

In transgenic cow both the alleles of the gene encoding normal form of prion protein (PrPC)
were knockout by the insertion of an antibiotic-resistant gene into the coding sequences. The
genetically modified animals were found normal for a variety of morphological and
physiological features including mental status, sensory and motor functions, immune
function, and brain tissue morphology. When brain tissue homogenates were collected from
wild-type and PrPC knockout cattle and incubated with brain homogenates of BSE-infected
cattle carrying the abnormal version of the prion protein, PrPBSE. Propagation of PrPBSE could
not be detected in the homogenates of PrPC knockout animals while it was readily detected in
the wild type homogenates. It suggests that the genetically engineered PrP C knockout cattle
could be resistant to BSE infection.

Mastitis: Mastitis is an infection of mammary glands of the cow. It can block milk ducts,
reduce milk output, and can also contaminate the milk with pathogenic microbes. It is caused
by the bacterium Staphylococcus aureus. These infections are contagious and readily spread
in the entire herd.

In an attempt to create cattle resistant to mastitis, transgenic cows were generated that
possessed the lysostaphin gene from Staphylococus simulana. Lysostaphin is an enzyme that
specifically cleaves components of the S. aureus cell wall. A transgene consisting of altered
lysostaphin gene under the control of the bovine β-lactoglobulin promoter was introduced
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 into cow fibroblasts. The nuclei from these cells were then transferred to enucleated oocytes
and activated. Blastocysts were implanted into the uterus of cows and several calves were
subsequently born. Transgenic cows expressing this protein in milk provide immunity to
suckling against S. aureus infections.

8. Transgenic Poultry
Transgenes are injected into the germinal disc that contains the female and male pronuclei.
After the administration of DNA to a germinal disc, each egg is cultured in vitro until
formation of embryo. Subsequently, it is placed in a surrogate egg to produce a hatchling.
Despite the technical difficulties, some transgenic lines of chickens have been established.

Transgenesis could be used to improve the genetic makeup of the chickens with respect to
resistance to diseases; lower fat and cholesterol levels in eggs; and better meat quality. The
egg, with its high protein content, could be used as a source for pharmaceutical proteins. A
transgene can be expressed in the cells of the reproductive tract under control of the
ovalbumin promoter and regulatory elements. This can yield up to 1 g of recombinant protein
in the eggs. Transgenic chickens that synthesize monoclonal antibodies, growth hormone,
insulin, human serum albumin, and alpha interferon have also been created.

9. Transgenic Fish
Transgenes have been introduced by microinjection or electroporation into the fertilized eggs
of fishes such as carp, catfish, trout, and salmon. Enhanced growth rates, tolerance of
environmental stress, resistance to diseases and model for pollution monitoring are some of
the traits considered for creation of transgenic fishes.

Transgenic salmon with higher growth rate: Transgene was created consisting of the
promoter region and polyadenylation signal from the antifreeze protein gene of the ocean
pout and the growth hormone cDNA from Chinook salmon. These transgenes were injected
into eggs of Atlantic salmon and Aqa Advantage salmon were created. Presence of promotor
from the antifreeze gene resulted in expression of growth hormone in cold waters. Hence the
transgenic salmon were larger and grew faster than the nontransgenic fishes (Fig. 11).
Conceptually, the faster growth of farmed salmon would lower the cost of the feed and lessen
the pollution of coastal waters. On 25 November 2013, Environment Canada approved the
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 product for salmon egg production for commercial purposes in Canada. In May 2016, the
Canadian Food Inspection Agency approved the sale of the GM fish.

Fig. 11: Transgenic Atlantic salmon overexpressing growth hormone (GH) gene. It shows accelerated rates of
growth compared to wild strains and nontransgenic domestic strains.
Source: https://alchetron.com/AquAdvantage-salmon-1733056-W

GloFish the first GM pet: Scientists at Yorktown Industries of Austin, Texas, created the
GloFish, a transgenic strain of Zebrafish (Danio rerio) containing a red fluorescent protein
gene from sea anemones. GloFish fluoresce bright pink when illuminated by ultraviolet light.
It was marketed as first „GM‟ pet in the United States. A variety of different coloured
GloFish are currently available at the pet stores (Fig. 12).

Fig. 12: Fluorescent transgenic zebrafish marketed as GloFish I the pet shops
Source: https://en.wikipedia.org/wiki/GloFish
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 10. Summary
 Transgenic animals has wider applications in the field of research, production of
pharmaceuticals and therapeutics, development of disease resistant livestock, production
of environment friendly animals and improving nutritional quality of the animal products.

 Transgenic mouse models have been developed for human genetic diseases, such as
Alzheimer disease, Huntington disease and many others.

 The knockout mice provide critical tools for understanding gene function and the genetic
basis of human diseases. Knockout Mouse Project (KOMP) was initiated in 2006 with the
goal of producing knockout mutation in each gene of the mouse genome.

 Transgenic cattle can be used for production and secretion of pharmaceuticals and
therapeutics in their milk. Human protein antithrombin produced in transgenic goat‟s milk
has been approved by U.S. Food and Drug Administration for the use in the individuals
with a hereditary deficiency for this protein.

 XenoMouse is transgenic mouse, produces fully human antibodies. First fully human
monoclonal antibody, produced in the XenoMouse (Panitumumab), has received
regulatory approval for use as a treatment for advanced colorectal cancer.

 Transgenic animals can produce organs for xenotransplantation to human beings. This
may be achieved by inserting human complement inhibitor gene or deleting α-Gal epitope
by knockdown the gene encoding 1,3-α-galactosyltransferase in the transgenic animals.

 Transgenic animals with improve nutritional quality has been produced. Increase casein
quantity, secretion of the enzyme lactase in the milk and knockout of β-lactoglobulin gene
are some of the desirable trait in the milk. The gene for enzyme desaturase from C.
elegans is expressed in transgenic pigs. This gene can convert omega 6 fatty acid into
unsaturated omega 3 fatty acid.

 Enviropig is environment friendly pig. It contains a transgene for the enzyme phytase. The
enzyme phytase is secreted in the saliva and digest phytate present in the feed. This
decreases phosphorus content in the excreta of the transgenic pig.
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ZOOLOGY Molecular Genetics

Transgenic animals: Applications
  Transgenic medaka containing gene for gfp can be used to monitor estrogenic compound
in the water bodies.

 Transgenic animals, resistance to infectious diseases such as Mastitis (mammary gland

abscesses), bovine spongiform encephalopathy (mad cow disease) and neonatal scours
(dysentery) have been developed.

 Transgenesis can be used to improve the genetic makeup of the chickens with respect to
resistance to diseases; lower fat and cholesterol levels in eggs and better meat quality.
Pharmaceuticals can also be produced in the egg of transgenic poultry.

 AquaAdvantage salmon contains a transgene construct with promotor of antifreeze gene

from ocean pout and growth hormone sequence of chinook salmon. These salmons are
larger in size and grow faster than nontransgenic salmon.

 GloFish is first GM fish, containing gene for fluorescent protein, Different types of
GloFishes are available at the pet stores.

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ZOOLOGY Molecular Genetics

Transgenic animals: Applications