Prospects for improving the quality or composition of animal products by creating transgenic animals.

Creation of transgenic animals opens real prospects for improving the quality or composition of animal products. For example, it is possible to reduce lactose in milk by creating transgenic cows and sheep, which have specific for mammary gland promoter (region of DNA to which RNA polymerase binds to start the synthesis of mRNA), linked to the gene lactase. Thus in cow (sheep) milk lactose can be cleaved into glucose and galactose. Such milk could be used in nutrition of newborn children suffering from hereditary lactose intolerance. For these children during infancy milk should be given only after processing by enzyme. In addition, milk would be useful in a variety of gastrointestinal human diseases associated with decreased activity of lactase (beta-galactosidase). The presence in the milk of various microflora caused problems associated with the storage, processing, consumption of milk and animal health. In this regard, the genes which are responsible for the production of antibodies against specific pathogens (RDBremel et al., 1989; UH Weidle et al., 1991). An important task is getting milk and dairy products containing thermostable enzyme lysozyme are constructed. During pasteurization of milk this enzyme, which has strong antibacterial property does not lose its activity, which will significantly increase shelf life of milk and products derived therefrom. In the acidic environment of the gastrointestinal tract lysozyme is inactivated. The possibility of the introduction of genes encoding the antibodies with protective effects against agents of cows mastitis are considered. Institute of Cytology and Genetics SD of RAS (Novosibirsk) and the Institute of Molecular Genetics, RAS (Moscow) established genetic construction pGoatcasGMCSF, which contained the regulatory region of the gene alpha-S1-casein goats carrying the human gene of granulocyte-macrophage colony-stimulating factor -GM-CSF (I.A.Serova et al., 2011). In the experiments, the injection of recombinant DNA into pronuclei of zygotes 4 transgenic mice were obtained. PCR shows the tissue specificity of expression of human GM-CSF only in the mammary gland of lactating females. Because mentioned construction is tissue-specific, it falls under the regulation of physiological signals of pregnancy and lactation.

Achievements of genetic engineering can be used to change the quality and yield of sheep wool. Further improvement of characteristics of sheep wool to a certain degree depends on the supply of hair follicles with nutrients necessary for their active functioning. The main obstacle is limiting supply of energy for the cell proliferation processes, and amino acids (lysine, methionine, arginine, histidine and cysteine) for the synthesis of keratin of coat fibrin. Enzymatic processes caused by rumen microflora, does not always completely provide the synthesis of amino acids, because at the splitting of proteins, most of their food goes to the synthesis of microbial self-proteins, which reduces the levels of important amino acids for hair growth. That is why priority of recombinant DNA technology, aimed at improving the performance of sheep wool, is to increase the efficiency of sheep’s feed utilization.

 In order to increase the amount of sulfur-containing amino acids (eg cysteine​​) which are needed for the biosynthesis of keratin proteins of sheep wool, Rogers GE (1990) it is necessary to create transgenic sheep, having in its genome bacterial genes coding for the synthesis of cysteine. These genes must be expressed only in the epithelium of the sheep’s gastrointestinal tract and determine the use of sulfur generated during of enzymatic processes of microorganisms. They obtained the first transgenic lamb, containing in its genome genes serinatsetiltransferaza (SAT) and O-atsetilserinsulfgidrilaza (OAS) of Salmonella typhimurium. K.A.Ward et al. (1990) used the SAT and OAS of Escherichia coli connected to the gene construct consisting of the promoter metallothionein-1a sheep, causing zinc-dependent expression. Research in this area continues. Animal selection for resistance to disease becomes more and more important. In contrast to vaccination, the effect of which is manifested in the lives of concrete individuals, genetically engineered immunity may be hereditary, which will elevate the line of agricultural animals that are resistant to certain infectious diseases. Resistance to a number of diseases are polygenic trait. For example, resistance to trypanosomiasis of certain African cattle are observed against their heat endurance and undemanding to of feeding and maintenance conditions. However, the resistance of the organism may be based on single genes. For example, resistance to diarrhea in newborn piglets or resistance to influenza in mice.This phenomenon was the basis for creation of transgenic animals that will probably develop immunity to certain infectious diseases. In this area of genetic engineering investigations of gene-specific antiviral effect of monoclonal antibodies brought to the forefront. The mechanism of action of monoclonal antibodies must be to a certain extent similar to the specific anti-immunization of animals. Transgenic mice were obtained that produce antibodies against specific antigens without preliminary immunization or contact with infection (Storb U., 1987). Genes of the light and heavy chains of monoclonal antibodies against 4-hydroxy-3-nitrophenyl acetate were integrated into the genome of rabbits and pigs (U.Weidle et al., 1991). The titers of specific antibodies in the serum of transgenic animals reached, respectively, 100 and 1000 mg / ml. There are data about obtaining of transgenic mice, sheep and pigs with gene constructs encoding the alpha-and khi-chain of antibodies against phosphorylcholine (D.Lo et al., 1991). The authors observed high levels of mice IgA, but only in transgenic mice and pigs. Bremen, and others since 1991 have identified and cloned Mx gene of mice which is responsible for immunity to influenza A virus, and are working on getting transgenic pigs on the bases of using this gene. The possibility of obtaining transgenic animals with increased concentration of lactoferrin in the mammary gland, in order to improve resistance to mastitis. Work is underway to obtain animals having in its genome the transgene of antisense RNA. Expression of anti sense RNA in the cells leads to hybridization with sense RNA, resulting in suppression of viral replication gene. Thus, by Russian researchers (TI Tikhonenko, MI Prokofiev., LK Ernst., 1991) gene of antisense RNA against adenovirus was constructed and transgenic rabbits were obtained. Animal cell lines (cell culture from the kidney), having a transgene showed high resistance against adenovirus as compared with the control cell lines. They have demonstrated the resistance of animals with transgenic antisense RNA against bovine leukemia at the organism level by contamination with the virus – causative agent of the disease. Thus, in transgenic rabbits with the mentioned genome titer of antibodies against p24 antigen was significantly lower (1:500) than in control animals (1:8000). The possibility of creation of intracellular immunization against certain viruses was demonstrated. Transgenic chickens were obtained, whose cells express leukemia virus capsid protein, which contributed to their stability to the disease.

The existence of breeds with genetic resistance to bacterial infectious diseases - mastitis (cows), dysentery (newborn piglets), cholera (poultry) is well known. If the basis of resistance to each of these diseases lying one gene, it is possible to create transgenic animals bearing it. At present, control of infectious animal diseases are provided by using vaccines and drugs. The cost of all these measures can reach 20% of the total cost of the final product.

For breeding animal lines resistant to infectious agents, it is possible to use another approach consisting in creating transgenosis of inheritable immunological mechanisms. From this point of view, a variety of genes responsible for the immune system: major histocompatibility complex genes, T-cell receptors, lymphokines is considered. To date the most promising preliminary results were obtained by introduction in mice, rabbits and pigs genes encoding H-and L-chain of a monoclonal antibody. The idea of this approach is to supply transgenic animal with inheritable defense mechanism, eliminating the need for immunization through vaccination.

Introduction into the recipient antibody genes, which bind to specific antigens, was called immunization in vivo. To do this, the genes of H-and L-chains of mouse monoclonal antibody immunoglobulins binding to the 4-hydroxy-N-nitrophenyl acetate, was administered by microinjection into fertilized mouse, rabbits and pigs oocytes. In all cases, in the serum of transgenic animals corresponding activity of the monoclonal antibody was detected. However, the number of monoclonal antibodies, containing chains of H-and L-, was very low. In order to establish whether it is possible to solve this problem,it is necessary to test different transgenic construction. The possibility of including in organism’s cells the genes responsible for synthesis of proteins of great importance in human and veterinary medicine, formed the basis of the strategy of transgenic animals as bioreactors. To this day most of these proteins are extracted from the tissues and biological fluids of man. For example, a clotting factor, interferon, alpha-1-antitrypsin, and other proteins are prepared from blood, growth hormone - from the pituitary gland. They are produced in small quantities because of the high cost and difficulty of extraction of human tissues. In addition, they may be contaminated with pathogens such as Hepatitis, AIDS, etc.

Transgenic animals used for the production of valuable biological products have several advantages over microorganisms-producers, as well as cellular systems. In simple recombinant systems, of microorganisms glycosylation, B-hydroxylation or carboxylation of mammalian proteins in most cases it is impossible or possible, but with insufficient accuracy. This changes the structure of proteins, which can not but reflect on their biological activity. Along with this, in drugs which are used by humans for therapeutic agents admixture of bacterial proteins is undesirable. The main disadvantage of genetically engineered cell culture is the low yield of protein. Industrial reactors used for the cultivation of producer cells, are expensive, both in terms of their value, and in respect of their service. Creation of transgenic animals also requires more resources and moreover it is not easy, but once bred line of such animals can produce a large number of proteins with low cost, which will pay back all the expenses for a short time. Production of biologically active human proteins from transgenic agricultural animals guarantee their environmental cleanliness, which practically comes to exploitation of animals-producers.
Foreign proteins can be synthesized by most tissues of the animal. Transgene expression in certain organs can be achieved by a combination of structural genes with specific regulatory elements. Significant advances in the production of animals-bioreactors were achieved in epithelial cells of the mammary gland by targeted transgene expression. Structural gene linked to a promoter milk protein gene (casein, laktoalbumin, lactoglobulin), in the first place will be expressed in the cells of mammary gland. It allows to receive useful products with milk. The choice mammary gland as a site of production of foreign proteins justified by its huge protein productivity. The total content of milk protein, depending on the animal species varies between of 2-10%, ie at 20-100 grams per liter. For commercial production of proteins with pharmaceutical importance, already enough one or more grams of recombinant protein. The most effective "bioreactor" is cattle which can provide about 35 grams of protein per 1 liter. If the purification efficiency will be 50% in this case 50 kg of protein will be received in the year from 20 transgenic cows. Figuratively speaking, two cows is enough in order to completely satisfy the annual requirement for protein C, which is used to prevent blood clots, and Factor IX - (Christmas factor) the cascade mechanism of blood clotting. To date, a number of recombinant proteins is known, such as human protein C, antihemophilic factor 1X, alpha-1-antitrypsin, tissue plasma activator, lactoferrin, human serum albumin, interleukin-2, urokinase, chymosin, etc., obtained from the milk of transgenic animals . Works on the production of these proteins, with the exception of alpha-1-antitrypsin and chymosin, are at the level of laboratory research and have not reached a stage which would be of commercial interest. One of the goals of transgenosis of cattle is changing in the milk the contents of various its components. Thus, the amount of cheese produced from the milk is directly proportional to its casein, so it's promising to increase the number of milk casein by hyper transgene expression of this protein.. In 1992, British scientists received transgenic sheep - producers of the human alpha-1-antitrypsin, which were used to treat people with emphysema. This medication is usually obtained exclusively from the blood (1 g of alpha-1-antitrypsin costs 110 USD). In four heads of the protein concentration was in the range of 1 g / l and in one it reached35 g / l, which corresponds to half of all the proteins in milk. At this level of production one sheep during a year will give so much protein as needed for treating 50 patients. Russian scientists (L.K.Ernst, G.Brem, M.I.Prokofev, I.L.Goldman etc.) got transgenic sheep secreting with milk chymosin enzyme at a concentration of mg/1l 200-300. Chymosin - the main component in the production of cheese obtained from abomasum of dairy calves and lambs. Thus the cost of chymosin derived from a new source, will be cheaper by 5-10 times. According to calculations of the authors from three liters of milk of transgenic sheep, it is possible to receive the quantity of enzyme, which is sufficient for the production of one ton of cheese from cow's milk.
Transgene expression in the cells of the mammary glands of sheep and goats does not have any adverse effects nor the females during lactation or the suckle offspring. In contrast, after introduction to pigs transgene of bovine growth hormone under the control of metallothionein promoter unfavorable effects were observed. In the group of transgenic pigs number of hormones varied, but in general the whole group more quickly put on weight. Unfortunately, this positive result partially devalued by different pathologies: animals showed ulcers, kidney failure, lameness, inflammation of the pericardium, reduction of joint mobility, susceptibility to pneumonia. The cause of these symptoms is not known.

Perhaps they are related to long-term presence in the body excess of growth hormone. In these experiments, the transgene was synthesized more or less continuously. Transgenic sheep with an increased rate of growth of wool were also created. For this cDNA of sheep insulinlike growth factor I was placed under the control of the murine keratin gene promoter with a high sulfur content, which provided the hyper-expression of cDNA. In this case, in transgenic sheep in contrast to pigs no adverse side effects were observed. Positive results were obtained in experiments with transgenic pigs. For example, healthy transgenic pigs were created, in the genome of which the following genetic structure was presented: the regulatory region of gene human beta-globin, two genes of alpha 1 human globulin and one gene of human beta A- globin. As a result of its expression in blood of pigs human hemoglobin was synthesized, in this case of replacing the promoter of human beta-globin gene with swine human hemoglobin was synthesized in much larger quantities. Human hemoglobin produced by transgenic pigs having the same chemical properties as the natural human. It can be cleaned from pig by ordinary hemoglobin chromatography.

These results indicate the possibility of replacing of whole blood used in transfusions with human hemoglobin, obtained by transgenosis. However, isolated hemoglobin carries oxygen is not as effective as hemoglobin in the red blood cells. Moreover, it is rapidly destroyed in the body of animal and its breakdown products are toxic to the kidneys. Thus, obtaining a substitute for human blood by means of transgenosis is a long way off.

Recently, much attention is paid to the use of animal organs for transplantation to man. The main problem of interspecies transplantation is hyperacute rejection. Hyperacute rejection involves the binding of antibodies to the host carbohydrate antigenic determinant on the surface of cells of transplanted organ. Antibodies cause acute inflammatory response (activation of the complement cascade) that is why mass death of cells bearing antibodies is occured and rapid loss of the transplanted organ is observed. In natural conditions inflammatory response is blocked by special proteins on the surface of cells lining the blood vessel walls. These proteins - complement inhibitors are species specific. It has been suggested that if the donor animals carried one or more genes of the human protein that inhibits the complement, the transplanted organ would have been protected from the primary inflammatory response. For this purpose the transgenic pigs were obtained carrying different human complement inhibitor genes. The cells of one of these animals were completely insensitive to the components of complement system. Preliminary experiments on transplantation of transgenic pig’s organs to primates have shown that tissue of transplanted organ is damaged weaker and it does not rejected a little longer. Perhaps transgenic pigs carrying the human complement inhibitor gene and deprived of basic pig cell surface protein, which causes acute rejection, provide a source of organs for transplantation to man.

 The first work on getting transgenic animals - producers of interleukin-2 turned out encouraging. Interleukin-2 being a soluble factor of T-helper lymphocytes involved in cell proliferation and differentiation of T-cell killer, plays an important role in ensuring the required level of immunity. Using a gene construct consisting of rabbit beta-casein DNA and structural human interleukin-2 gene, rabbits were obtained secreting with milk active form of the protein.
Thus, integration of one or more genes in mammalian embryos is achieved and their expression as well as the transmission to the offspring is proved. However, the difficulties and uncertainties should be emphasized with which still related technique for producing transgenic animals. Mechanism of integration of the gene in mammalian cells is still poorly understood. This integration occurs randomly and not connected with a specific region of a chromosome. Another difficulty is due to the instability of the cells in which gene (s) is introduced: it may be lost or modified as a result becomes inactive. Finally, the activity of genes is determined not only by sequences of nucleotides that provide gene transcription with the formation of mRNA, but as well as other sequences of nucleotides, which are often far from their own gene. These sequences are administered with a gene to achieve full expression of the it. For example, the gene responsible for the synthesis of alpha-globulin, is regulated by a DNA sequence located in front of it.

The results achieved in the field of genetic engineering on getting transgenic mammals allow to deepen our knowledge about gene expression that in the future facilitate gene transfer and identification of factors that contribute to a more complete expression of the genetic information stored in transgene. In addition, insertion of a foreign gene in the section of the genome of cell, where normally is located homologous gene, may open the way for the treatment of genetic diseases, as this would replace.


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