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
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 Principal Investigator : Prof. Neeta Sehgal
Department of Zoology, University of Delhi
Development Team
ZOOLOGY Molecular Genetics
Transgenic animals: Applications
2 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
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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
ZOOLOGY Molecular Genetics
Transgenic animals: Applications
4 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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Transgenic animals: Applications
5 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
6 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
Transgenic animals: Applications
7 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.
ZOOLOGY Molecular Genetics
Transgenic animals: Applications
8 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
ZOOLOGY Molecular Genetics
Transgenic animals: Applications
9 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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Transgenic animals: Applications
10 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.
ZOOLOGY Molecular Genetics
Transgenic animals: Applications
11 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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Transgenic animals: Applications
12 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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13 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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14 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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Transgenic animals: Applications
15 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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16 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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17 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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18 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.
