Dairy At Glance

Transgenic livestock: Principles, Methods and Potential

Applications in Reproductive Biotechnology

S.D. Kharche*, Parul Yadav and A.K. Goel

Central Institute for Research on Goats, Makhdoom, Farah-281122, Mathura (U.P)

 

Transgenic animals are animals that have been genetically modified, through the introduction of foreign DNA or RNA, so that they overproduce, under produce, lack the production of, or have modified expression of particular protein. Transgenic animal may be defined as one whose genome has been permanently altered by the addition, deletion or modification of specific genes. The term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981). Transgenic animals were first produced nearly two decades ago. Most transgenic animals studies involve mice, other transgenic species, such as rabbits, rats, hamsters, sheep, goat, swine, Often mice are not suitable transgenic models, for example in some human genetic disorders or for the production of large quantities of biopharmaceuticals, where larger more closely related species would be more effective.

          During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two different embryos of different strain were combined together at an early stage of development (eight cells) to from a single embryo that subsequently developed into to chimeric adult, exhibiting characteristic of each strain. This technology allows the transfer of genes of interest from on species to another, thus permitting genetic improvements as well as better understanding of how genes function with an individual. Transgens can also be expressed in a specific tissue at a given time or at a particular stage of development. Moreover, transgenic offers the potential for great genetic advances in livestock production through improvements in animal health and production traits such as growth, milk, meat & wool production.

          Transgenic animal system combines the virtues of cells culture and congenic breeding strategies while avoiding the negative aspects of each system. Using transgenic techniques, a characterized genetic sequence may be evaluated with in the specific genomic background of whole animal. Genes can be transferred across species boundaries and can be modified to function very differently than they do in their native have application in both food production & biomedical arena. As efficiencies of this technology improve, other species of transgenic will become more commonplace and form (gene products, tissue specificity & timing of expression can be altered). The ability to redirect expression of genes to another organ has spawned the transgenic bioreactor industry. For the most part, transgenic bioreactors are farm animals designed to produce new proteins in their milk or other body fluids. It is envisioned that this approach will enhance our lives through their contributions to medicine & agriculture

Techniques for producing Transgenic livestock

          Several methods can be use to introduce exogenous DNA into developing mammalian embryo in such a way that it may ultimately become stably integrated into a chromosome of the resulting animal. There are five general methods for production of transgenic animals: Pronuclear injection of DNA microinjection, viral vectors, gene targeting in embryonic stem cells, sperm medicated gene transfer and biolistics. 

(a). Pronuclear injection: -

          This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of same species or from a different species, into pronucleus of a fertilized ovum. Fertilized pronuclear stage embryos are flushed from the oviducts of species of interest. One or both pronuclei are injected with 1 or 2 picolitres of DNA construct (1 or 2 µg/ml) just until the pronucleus visibly swells. Volume is enough to deliver from 100 to 1000 copies of transgene. Pronuclear swelling is also thought to induce chromosomal breakage, which improves chances of transgene incorporation. Injected embryos are either surgically transferred to recipients or culture for a period of time then transferred to recipients. The percentage surviving that are transgenic is even lower and varies for different species, averaging ≈5% for laboratory animals (mice, rats, & rabbits) & ≈ 1% or less for farm animals (cattle, sheep, goats & pigs).

(b). Viral vectors

            Viral vectors are another mode for introducing foreign genes into animals. They are not used as frequently as pronuclear injection due to complexity of generating replication deficient vectors and the species specificity of these vectors. Viral vector are however the method of choice for chickens as well as gene therapy protocols. The two most prevalent types of viral vector utilized for producing transgenic animals are as follows.

(i). Retroviral vectors: Retroviruses are single stranded RNA viruses that infect dividing cells in a species-specific manner. Once inside the host, the RNA virus is reverse transcribed to DNA, which can than be incorporated into the host genome.

(ii). Adenoviral vectors: Adenoviruses are DNA viruses that infect only non-dividing cells. These viruses once within the host cell do not incorporate into host chromosome but rather replicate episomally (independent of chromosomes).

(b). Gene targeting in embryonic stem cells

          Gene targeting is another powerful tool for producing genetically modified animals. This procedure involves genetically modifying embryonic stem cells (ES) cells through homologus recombination for the production of transgenic animal. This procedure allows for complete loss of gene function (gene knockout), subtle modification of genes, insertions, deletion or even translocation making this technique incredible powerful in field of transgenesis.

(i). Introduction into ES cells: Embryonic stem cells are unique cell lines derived from inner cell mass of early preimplantation embryos. ES cells can be genetically manipulated in culture, selecting for those that have incorporated transgene properly and than reintroduced into host blastocyst stage embryos by microinjection.

(ii). Homologous Recombination: Two phenomenon drive this recombination reaction: regions of complete homology and double-strand breaks within the DNA. This method is very inefficient, yielding in 10targeted events, so it is not practical for use on embryos.

(iii). Site-specific Recombination: The most recent advancement in gene targeting is the use of site-specific recombination system. Two of systems being utilized are Cre lox-p and Frt flipase. Site-specific recombination offers greater potential with ES cell technologies. This could be useful for production of biopharmaceuticals in milk because a mammary specific gene can be tagged with a Lox-P site.

(c). Sperm mediated gene transfer

          An innovative approach to production of transgenic mice through sperm mediated gene transfer was described. In this case sperm cells were “Killed” by sperm methods-including freeze thawing: freeze drying and exposure to TritonX-100, before a rapid co-incubation with plasmid DNA following this procedure, sperm heads and DNA were co-injected into oocyte cytoplasm (ICSI). As many as 94% of the resulting mouse blastocysts, (ranging from 64 to 94%) and 17 to 21% of the resulting offspring were transgenic. Moreover 8 out of 11 founders were able to transmit transgene to their progeny. This method caused disruption of sperm membrane and damage of nuclear DNA in the form of single strand breaks. Both events are likely to facilitate association of exogenous DNA with nuclear structures which in turn stabilizes the transgene within oocyte, their by facilitating its integration in resulting embryonic genome.

(d). Biolistics

            The gene particle gun or biolistics is another option for introduction foreign DNA or RNA into cells. Small gold particals are coated with the transgene and are shot into the target tissue. Some of the transgene will be propelled into cytoplasm and way into nucleus. It has been utilized mainly in gene therapy and vaccines protocols, but some have used biolistices for introducing.

Applications of Transgenic livestock

There are list of purposes for which transgenic animals have been used which indicates the wide ranging application of this technology.

(1). Biomedical application of transgenic animals

(a). Gene pharming: The important use of transgenic animals involves the biological production of valuable human protein, enzymes, hormones and growth factors. Current technique in biotechnology industry use large-scale cell cultures to generate products in biological system. The use of transgenic animals, particularly large mammals as bioreactors (“pharmaceuticals pharming”) is cost effective alternative to cell culture methods. Those are more similar to human proteins than those produced by bacterial and yeast system.

          By targeting expression to mammary gland via use of mammary gland-specific promoter elements, large amounts of numerous heterologus recombinant proteins have been produced. In bovine mammary gland of transgenic cows at least two different pharmaceuticals proteins, in caprine mammary gland five proteins, in the ovine system four proteins, in the pig two and in transgenic rabbits seven proteins have been produced. These could be purified from milk of transgenic rabbits, sheep, goat and cattle. Human lactoferrin has recently been reported to be produced in the large amounts in mammary gland of transgenic cows. Products such as anti-produced III (AT III), & α -anti-trypsin (α - AT) or tissue plasminogen activator (tPA) are currently in advanced clinic trials. Human clotting factor VIII cDNA construct can be expressed in mammary gland of transgenic mice, rabbit and sheep. Transgenic animal for pharmaceutical production should (i)-Produced the desired drug at high levels without endangering its own health (ii)-Pass its ability to produce the drug at high levels to its offspring.

(b). Xenotransplantation: Xenotrasplants are transfer of organs or tissues between discordant species from animals to human.

(i). Transplantation of solid organs: Currently a group of people is living only because of transplantation of an appropriate human organs (e. g. all transplantation. The pigs seems to be optimal donor animal because-

u  Pigs are a domesticated species.

u  Pigs grow rapidly.

u  Pigs have short reproductive cycle and large litters.

u  Organs have a similar size as human organs.

u  Porcine anatomy and physiology are too different from those in humans.

Transgenic pigs that show high expression of hcD59 predominantly in heart, kidney and pancreas but also other target organs were identified and transgenic lines established. Transgenic endothelial cells and fibroblasts were protected against complement mediated lysis. Perfusion studies using isolated porcine kidneys employing human blood revealed a significant protective effect against human antigenic response. Another promising strategy towards successful xenotransplantation is the knockout of antigenic structures on surface of the porcine organ. These structures are known as 1,3α-epitopes are produced from gene for 1,3 α -galactosyltransferase. Recently the generation of piglets in which one allele of the α -galactosyltransferase locus has been knockout was reported.

(ii). Use of xenogenic cells and tissue: Another promising area of application for transgenic animals will be the supply of xenogenic cells and tissues. Xenogeic cells, in particular from the pig hold great promise with regard to successful cell therapy for human patients. These cells provide several significant advantage over the other approaches such as implantation at the optimal therapeutic location (i.e. immuno privileged sites as brain), possibility for manipulation prior to transplant to enhance cell function, cry preservation, combination with different cell types in some graft. There are already numerous examples for successful application of xenogenic cell therapy.

u  Porcine islet cells have been transplanted to diabetic patients and were shown to be at least partially functional over limited period of time.

u  Porcine fetal neural cells were transplanted into brain of patients suffering from Parkinsons disease and Huntingtons disease.

u  The potential uses of neural cells are stroke and focal epilepsy.

u  Human, foetal neural cells have also been employed as transplants into parkinsons and Huntingtons disease patients.

u  Olfactory ensheathing cells (OECs) or Schwann cells derived from hcD59 transgenic pigs promoted axonal regeneration in rat spinal cord lesion thus cells from genetically modified pigs may serve as therapeutic measure to restore electro physiologically functional axons across the site of a spinal cord transaction.

u  Xenogenic porcine cells may also be useful novel therapy for liver disease. Upon transplantation of porcine hepatocytes to Watanabe heritable hyperlipidenic (WHMHL) rabbits (a model for familial hypercholesterolemia) the xenogenic cells migrated out of the vessels and integrated into the hepatic parenchyma. Thus integrated hepatocytes provided functional LDL receptors and thus reduced cholesterol level by 30-60% for at least 100 days.

u  A clone of bovine adrenocortical cells restored adrenal function upon transplantation to adrenalectomized SCID mice. This finding shows that functional endocrine tissue can be derived from a single somatic cell.

u  Xenotransplantation of retinal pigment epithelial cells hold promise to treat retinal disease such as muscular degeneration which is associated with photoreceptor losses.

u  Porcine or bovine fetal cardiomyocytes or my oblast may provide a therapeutic approaches for the treatment of ischemic heart disease. Similarly xenogenic porcine cells may be valuable of the repair of skin or cartilage damage.

In light of emergence of more efficient protocols for genetic modification of donor pigs and new powerful immunosuppressive drugs, one can expect xenogenic cell therapy to evolve as an important theraptic option for the treatment of human disease.

(c). Blood replacement: Functional human antibody has been produced in transgenic swine. The transgenic protein could be purified from the porcine blood and showed O2 binding character similar to natural human antibody. The main obstacle was that only small proportion of R.B.C. continued the human form of Hb. Alternative approaches to produce human blood substitutes have focused on chemical cross-linking of Hb to superoxide dismutase system.

(d). Production of a new class of antibiotics: Cationic anti-microbial peptides with increasing antibiotic resistance in bacterial species, there is a growing need to develop new classes of anti-microbial agents. Cationic anti-microbial peptides (AMP) have many of the desired features because they possess a broad spectrum of activity, kill gram-positive and gram-negative bacteria rapidly, are unaffected by classical resistance genes and are active in animal modes. AMPs belong to the innate immune defense, which acts as a first barrier ahead of humoral and cellular immune systems, and neutralizes bacteria by interacting specifically with their cell membranes. AMPs from livestock species would be superior anti-microbial drugs because they would lack cytotoxic effects that were found for insect peptides; the evolution of resistance would not affect the human specific innate immunity Recombinant production will keep the production costs low. Goat, sheep and other farm animals can be exploited for the production of new class of antibiotic by using transgenic approach.

(e). Development of human genetic disease models: Another area where transgenic animals, especially pigs, will have a significant impact on society will be in the development of human genetic disease models. A number of genetic disease models have been generated in mice for atherosclerosis, sickle cell anemia, Alzheimer’s disease, autoimmune diseases, lymphopoiesis, dermititis, and prostate cancer. These models for the most part require “knocking out” the function of a gene or replacing an existing gene with a mutant form. Many of these models will have been replicated in farm animals to be useful. Unfortunately, the stem cell technology required to generate most of the disease models is still in development for livestock.

(2). Transgenic animals in material industry

          A very novel approach to produce useful fiber has been recently accomplished using the milk of transgenic goats. The Canadian biotech company Nexia has bred transgenic goats to produce “Biosteel” in their milk. This is spider silk protein, stronger and more flexible than steel, which could be used for bullet proof vests and in the aerospace industry. Another advantage of spider silk is that is compatible with human body therefore the material could also be used to help tissue repair, wound healing and to create super thin biodegradable sutures for eye of neurosurgery. Nexia used embryos extracted from does, transfixed with foreign genes and re-implanted to ordinary does as surrogate mothers. The protein monomers can be assembled in the laboratory or factory to produce fibers with properties approaching those seen in the natural spider milk.

(3). Transgenic animals in agriculture

          Areas of transgenic technology in agriculture being heavily pursued include improvements in milk quality, improvements in viral resistance and natural immunity and improvements in production traits such as wool and carcass characteristic.

(a). Improved milk quality: Modification of milk protein composition is possible through transgenic animal technology. Improvement in cheese production may be made through increasing casein expression. This will improve thermal stability, thus improving curd formation. Changes in phosphorylation of casein would also allow for cheese to be made either softer or harder. By replacing animal milk protein genes with human genes, better milk substitutes could be generated for infants. With an estimated 90% of adults having lactose intolerance, modification of this sugar or its cleavage to glucose and galactose would provide a useful alternative. Lysozyme concentration is 3000 fold higher in human milk than in that of cows. This protein is effective against bacteria causing food spoilage and food-borne disease. These antibacterial protein genes may also reduce incidence of mastitis, a disease that results in large financial losses to diary industry every year.

(b). Improved disease resistance: Another means by which transgenic animals may benefit animals in agriculture is though improved disease resistance. Several groups are taking different approaches to enhance animal’s immune response, including transfer of genes for natural immunity, antibodies and viral proteins. Natural immunity to disease may eventually be transferred from one species to another, as genes regulating disease resistance are determined. Several studies have utilized the Mx-1influenza viral resistance gene in mice and pig transgenesis with variable results.

Transfer of performed antibodies is another approach for improving disease resistance.

Transgenic mice, rabbits, sheep and pigs produce light and heavy chain components of antibodies. Mice have been produced that have antibodies towards the bacterial surface antigen phosphorycholine.

Researches have also produced transgenic sheep and mice that produce the env gene of visna virus and chickens, which express avian leucosis viral envelope proteins. These transgenic animals produced antibodies to these proteins, which may improve resistance to disease by blocking the entrance of virus into the cells.

(c). Carcass composition: Transgenic pigs bearing a human metallothionein promoter/porcine growth hormone gene construct showed significant important trait a growth rate, feed conversion and body fat/muscle ratio without pathological phenotype known previous growth hormone construct. Similarly pigs transgenic for human insulin IGF-1 had 30% lean mass, 10% more carcass lean tissue and 20% less total carcass fat. The commercialization of these pigs has been postponed due to current lack of public acceptation of genetically modified foods. Recently an important steps toward production of more healthful pork has been made by creation of first pig transgenic for spinach desaterase gene that produce increased amounts of non-saturated fatty acid. The pigs have higher ration of unsaturated to saturated fatty acid in strieated muscle, which means more healthful meat since a diet rich in non-saturated fatty acid to be correlated with a reduced risk of stroke and coronary disease.

(d). Wool production: Transgenic pigs carrying a Keratin IGF-1 construct showed that expression in skin and fleece was about 6.2% greater in transgenic than in non-transgenic animals. No adverse effect on health or reproduction were observed. Approches designed to alter wool production by transgenic modification of cystein pathway have met with only limited success, although cystein is known to be rate limiting biochemical factor for wool growth.

(5). Pollution free environment

       Phytase pigs have been developed to address problem of manure related environmental pollution. These pigs carry a bacterial phytase gene under the transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. Without bacterial enzyme, phytate phosphorus passes undigested into manure and pollutes the environment. With bacterial enzyme, fecal phosphorus output was reduced by up to 75%.     

Akshay Sadana

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Posted Date : 31/03/2015 Posted By : Admin