Transgene expression in the epithelial cells of mammary gland. The advantages of transgenic animals over microorganisms-producers of biopreparations
Gene transfer in farm animals can be used in improving the productivity and quality of animal products, increasing resistance to disease and the creation of transgenic animals - bioreactors of valuable biologically active substances. Ernst LK (1996) reported that in transgenic pigs with growth hormone gene the final body weight was 15.7% higher than in control animals. According to Brehm, et al (1991), in the offspring of transgenic pigs that received the corresponding modified feed ration with a high content of protein and an additional amount of lysine, had higher daily additional weights. In contrast to these results, there are cases of transgene expression without phenotypic effect. For example, in transgenic rabbits, pigs, and sheep, in none of the cases of expression of human growth hormone any phenotypic change was not observed. (R.E.Hammer et al., 1985). .G.Pursel et al. (1987) with the same gene obtained pigs with established expression of the gene, but without changing the rate of growth. These same researchers (1988, 1989) also does not recorded corresponding acceleration of growth in transgenic pigs with growth hormone gene. Transgenic sheep with growth hormone gene had its increased level, but did not differ from controls on the intensity of growth. However, transgenic pigs had a more than twofold reduction in the thickness of bacon, and fat content of transgenic sheep was about 4-5 times lower than that of in control counterparts. In this regard, a number of suggestions were expressed, one of which links the absence of a specific phenotypic effect in animals with poor recognition of heterologous hormone by its receptor molecules or with the post-translational modifications (KAWard, CDNancarrow, 1991). This suggestion soon gets experimental evidence in actively growing pigs, in genome of which additional copies of the own growth hormone gene was incorporated (VDVize et al., 1988). However, subsequent experiments on sheep gave a different result. Hopes that in the organism of sheep her own gene would "work" better than foreign, did not materialize. Transgenic sheep with high sheep growth hormone showed a slight increase in body weight and were physiologically abnormal. The difference in growth rate between transgenic mice and transgenic farm animals, some researchers explain as follows. Most pigs used in the experiments on the transfer of genes came from populations in which selection on the growth of productivity was carried out for a long time, whereas in strains of mice selection for this indicator was not conducted. Indeed, mice in the population of which selection was carried out by the growth rate within 30 generations had significantly lower body weight as compared with transgenic counterparts. Therefore, the introduction of foreign genes into a population of strains of mice which were not subjected to selection for growth rate, causing a jump to a higher level of growth.
Achievements of genetic engineering in the selection of animals resistant to diseases
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.
Genetic and cellular engineering are the most important methods (tools) underpinning modern biotechnology. Methods for Cell Engineering focused on construction of a new cell type. They can be used to reconstruct viable cells from the individual fragments of different cells, to combine whole cells belonging to different species with the formation of cells carrying the genetic material of both the original cells, and other operations. Genetic engineering methods are aimed at design of new combinations of genes that do not existing in nature. As a result of genetic engineering techniques recombinant (modified) RNA and DNA can be obtained. After a certain manipulation of these genes by introducing them to other organisms (bacteria, yeast and mammals), which having received the new gene (s) will be able to synthesize the final products with the changed, desired properties. In other words, genetic engineering makes it possible to obtain transgenic plants and animals.
People have always thought about how to learn to control nature, and searched for ways to get, for example, plants with improved properties: a high yield, a large and delicious fruit, or with increased cold tolerance. Since ancient times, the main method used for this purpose was selection. It is widely used to date, and it is aimed at creating new and improving existing varieties of cultivated plants and breeds of domestic animals, strains of microorganisms with valuable features and properties for a man. Breeding is based on the selection of animals (plants) with distinct symptoms and further favorable crossing of such organisms, whereas genetic engineering allows direct intervene in the genetic apparatus of cells. It is important to note that during the traditional breeding for obtain hybrids with the desired combination of useful features is very difficult because very large fragments of the genome of each parent are transmitted to the offspring, whereas the genetic engineering techniques allow to work more often with one or several genes. As a result, one or more of the useful features that is very valuable for the creation of new varieties and new forms of plants can be added, without losing other useful properties of plants. It became possible to modify in plants, for example, their resistance to climate and stress, or susceptibility to insects or disease widespread in some regions, drought, etc. There is considerable research to improve the nutritional value of various crops such as corn, soybeans, potatoes, tomatoes, peas, etc. Historically, "three waves" in the creation of genetically modified plants were distinguished. The first wave (late 1980s) is the creation of plants with novel properties of resistance to viruses, parasites, or herbicides. In plants of the "first wave" only one additional gene was introduced, and it was forced to "work" that is, to synthesize an additional protein. "Good" genes "were taken" from plant viruses (for the formation of resistance to this virus) or from soil bacteria (to form a resistance to insects, herbicides). The second wave (early 2000s) is the creation of plants with new consumer properties: oilseeds with a high content of reformulated oils, fruits and vegetables with high content of vitamins, more nutritious grains, etc. Today, scientists create plants of "third wave", which in the next few years appear on the market. They are plant vaccines, plant bioreactors for the production of industrial products (components for various types of plastics, dyes, industrial oils, etc.), plant –factory for medicines, etc.
Genetic engineering in livestock have another problem. Technology which is achievable at the present level is the creation of transgenic animals with a specific target gene. For example, transgenic goats, by the introduction of corresponding gene can produce a specific protein - factor VIII, which prevents bleeding in patients with hemophilia, or an enzyme - trombokinasa that promotes resorption of the thrombus in blood vessels, which is important for the prevention and treatment of thrombosis in humans.
Transgenic animals produce these proteins are much faster and much cheaper than the very method of the traditional. Another no less important direction of modern biotechnology is the "cell engineering", engaged in the acquisition of new cells with desired properties by merging the parental cells. Development of methods for somatic cell fusion made it possible for researchers to seek new ways of solving actual problems in medicine, veterinary medicine and agriculture. In particular, a new approach to obtain homogeneous (homogeneous) antibodies against infectious agents or other antigens that have great diagnostic value. A method for obtaining homogeneous of antibodies is called hybridoma technology.
Its essence is as follows. In the cause of entering organism specific foreign substance (antigen) protective body - antibodies which are heterogeneous in their physicochemical and biological properties are formed. Antibodies are produced by different lines of B-lymphocytes and they are directed to different regions (determinants) of antigen. If single cell of lymphocyte could be isolated and cultivated in vitro, then the resulting clone would have produced one type of antibody - a monoclonal antibody. However, lymphocytes can not grow outside the body. At the same time, there are cancers - myeloma cells that synthesize a large number of abnormal immunoglobulins. They are capable of unlimited growth and produce immunoglobulins which are similar in all respects. Thus, studies to obtain cell hybrids capable of producing monoclonal antibodies in vitro conditions have had a theoretical basis. At the end of 1974 hybridoma by fusion of myeloma cells and spleen lymphocytes from mice immunized with sheep erythrocytes was obtained by Koehler and Milstein. They were able to isolate clones of cells producing a specific type of antibody molecules and grow in the medium outside the body. Cell chimeras, called hybridomas, inherit the capacity for unlimited growth in culture and at the same time to produce antibodies of identical specificity, ie, monoclonal antibodies. The latest become a powerful tool in the development of efficient methods for diagnosis and treatment of diseases.
Another method of cell engineering is cloning which finds its application in animal husbandry. Cloning is a method of producing identical offspring by asexual reproduction. Otherwise, the cloning can be defined as the reproduction of genetically identical copies of a single organism. In nature, cloning is widespread among different organisms. In plants, natural cloning is done with various methods of vegetative reproduction in animals - in various forms of parthenogenesis and polyembryonesis (polyembryone from "poly" and Greek word embrion - «germ" - the formation of multiple embryos of animals (twins) from the same zygote as a result of an incorrect division due to the impact of random factors).
As for human example of polyembryony can serve the birth of identical twins, which are natural clones. There is a widespread clonal propagation among the crustaceans and insects.
The first artificially cloned multicellular organism was the sheep Dolly in 1997. The essence of the technique of "nuclear transfer" used in cloning is the replacement of cell nucleus of a fertilized egg to the nucleus extracted from the cell body, an exact genetic copy of which is scheduled to receive. To date currently developed not only the methods of reproduction of the organism from which the cell was taken but also those from which the genetic material was taken. There was potential to reproduction a dead body, even when it remain a minimum of parts - only necessary that one could isolate DNA. Cloning opens new prospects in the agriculture and animal husbandry. Animals with high productivity of eggs, milk, wool, or animals that produce the necessary enzymes to man (insulin, interferon, etc.). By combining the techniques of genetic engineering with cloning, we can derive transgenic agricultural plants that are able to protect themselves from pests or to be resistant to certain diseases. Here are listed just some of the opportunities opening through the use of this new technology. However, with all its advantages and prospects, so important for solving many problems of humanity, cloning is one of the most discussed areas of science and medical practice. This is due to the unresolved whole complex of moral and ethical and legal aspects of manipulation with sexual and stem cells, fate of embryonic and human cloning.
Cloning of organisms can be complete or partial. A full cloning recreates the whole organism as a whole, and the partial - recreated only certain tissues. Technology recreating the whole organism is extremely perspective in the case of the need to preserve rare species of animals or for the restoration of extinct species. Partial cloning - could become a major trend in medicine, because the cloned tissues can compensate for the deficiency and defects of the human body's own tissues, and, most importantly, they are not rejected during transplantation. Such therapeutic cloning did not initially involves getting the whole organism. Its development is consciously stopped in the early stages, and the resulting cells which are called embryonic stem cells (embryonic or fetal stem cells are the most primitive cells that arise in the early stages of embryo development, can develop into all cells of the adult organism) are used to generate the desired tissue or other biological products. Experimentally proved that therapeutic cloning can also be successfully used to treat certain human diseases, which are still considered incurable diseases (Alzheimer's, Parkinson's disease, heart attack, stroke, diabetes, cancer, leukemia, etc.), to avoid the birth of children with the Down syndrome and other genetic diseases. Scientists see the possibility of successful use of cloning techniques in the fight against aging and increasing longevity. The most important application of this technology is the area of reproduction, for example, case of sterility, both female and male.
In his lecture only a few of the many problems that arise in connection with the rapid development of biotechnologies and their invasion into human life are given. Of course, the progress of science can not stop and its problems poses faster than society can find answers to them. To cope with this situation we can only understand how is important discussing in society, ethical and legal issues that arise with the development of biotechnology. The presence of enormous ideological differences on these problems raises a serious need for consciously necessity of state regulation in this area.
The proposed discipline consider contemporary issues of Veterinary Medicine and Animal husbandry (immuno-and gene diagnostics, a new generation of vaccines, pharmaceuticals and food products, the creation of transgenic animals embrio- engineering, biotechnology, and biosecurity), which can be successfully solved using the techniques of modern biotechnology.
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