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ZOOLOGY Molecular Genetics

Transgenic animals: Applications

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Paper No. : 16 Molecular Genetics

Module : 17 Transgenic Animals: Applications

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

Department of Zoology, University of Delhi

Paper Coordinator : Prof. Namita Agarwal


Content Writer : Dr. Kamal Kumar Gupta,

Deshbandhu College, University of Delhi

Content Reviewer : Dr. Surajit Sarkar

Department of Genetics, South Campus, University of Delhi

Principal Investigator : Prof. Neeta Sehgal


Development Team



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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



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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



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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



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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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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.



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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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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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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

https://in.pinterest.com/pin/325736985531240468/



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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 PrPC 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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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.

input template epg pathshala - epgp.inflibnet.ac.in

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