Problems of genetic engineering in the creation of transgenic animals

⇐ ПредыдущаяСтр 6 из 6

For breeding of improved breeds of domestic animals and birds (cows with a higher milk yield, sheep with high-quality wool, chickens with higher egg production, etc.) many rounds of crossing and selection are conducted, each time using animals as producers with the best characteristics. As a result, eventually it is possible to get more or less clean lines of highly productive breeds of animals. Nevertheless strategy of crossing and selection, requiring more time and material costs, turned out extremely successful, and today nearly all aspects of the biological basis of developing new breeds of livestock can be brought to it.

However, after the effective genetic line is obtained, introducing new features by crossbreeding and selection becomes more difficult. Thus, the line with the new "valuable" gene can also carry "bad" genes, so that the descendants can be less productive. To ensure that the new, improved line will retain the original useful features and will acquire new ones, it is necessary to develop a completely new strategy. To create transgenic animals it is necessary to obtain such individuals, in which all or part of the sex cells have transgene. Investigations of transgenic animals and their offspring showed that, despite the introduction of DNA in the early stages (in the pronucleus of a fertilized oocyte) mosaic animals may appear. Mosaic are animals consisting of two or more cell lines originating from a single zygote, but with different genotypes. Such animals, besides cell lines with transgene have non-transgenic cell lines. Therefore, if the germ cells do not contain a transgene, offspring can not inherit a foreign gene from transgenic parent. It is established that about 30% of the primary transgenic animals are mosaics. Transgenic technology developed and refined on laboratory mice. Since the early 1980's. in different strains of mice hundreds of genes were introduced. These studies have contributed significantly to establishment of mechanisms of gene regulation and tumor development, the nature of immunological specificity, Molecular Genetics of growth and development, and other fundamental biological processes. Transgenic mice played their role in the investigation of the possibility of large-scale synthesis of drugs, as well as in creation of transgenic lines to simulate a variety of genetic diseases of man. The introduction of foreign DNA into mice was carried out by different methods: 1) by using retroviral vectors to infect cells of embryo in the early stages of its development prior to implantation of embryo into a female recipient, and 2) by microinjection into the increased sperm nucleus (male pronucleus) of fertilized egg, and 3) by introduction of genetically modified embryonic stem cells in pre-implanted embryo in its early stages of development.
The use of retroviral vectors. The advantage of the method based on the use of retroviral vectors, over the other methods of transgenosis consists in its efficiency. However, the insert size in this case is limited to 8 kilobases, so that the transgene may be deprived of the adjacent regulatory sequences required for its expression.

The use of retroviral vectors has another great drawback. Although these vectors are created so that they were defective in replication, the genome of a strain of retrovirus (helper viru, which is necessary to obtain a large number of vector DNA can enter the same kernel as the transgene. Despite all the measures taken, the helper retroviruses can replicate in the body of the transgenic animal that is absolutely unacceptable, if these animals supposed to use as food or as a tool to get a commercial product. And because there are alternative methods of transgenic, retroviral vectors are rarely used to create transgenic animals that have commercial value.

Microinjection of DNA. Currently for the creation of transgenic mice microinjection of DNA are most commonly used. It consists in the following. The work begins with the stimulation of hyper ovulation in female donors to increase the number of eggs in which foreign DNA will be injected. At first pregnant mare serum are injected to females and after about 48 hours human chorionic gonadotropin is administrated. As a result of hyper ovulation about 35 eggs are formed instead of usual 5-10. Then females with hyper ovulation is crossing with males after which they were sacrificed, fertilized eggs are washed out from the oviduct, and immediately DNA is injected into fertilized eggs. Introducing transgene design is often in a linear form and does not contain prokaryotic vector sequences.

In mammals, after the penetration of sperm into the oocyte nucleus of spermine (male pronucleus) and nucleus of the ovum exist separately. After nucleus of the ovum completes mitotic division and become female pronucleus nuclear fusion (karyogamy) may occur. Male pronucleus is usually much more than feminine, it is easy to localize it by using the sectional microscope and to introduce foreign DNA. Experienced experimenter can inoculate several hundred eggs for a day.

After the introduction of DNA 25 to 40 oocytes are implanted by microsurgical ways in "surrogate" mother whose in the false pregnancy state by mating with a sterile male. In mice, the pairing is the only known way to prepare the uterus for implantation. Since sterile male does not produce sperm, no oocytes of "surrogate" mother is fertilized. Embryos are developed only from introduced oocytes and pups are born in 3 weeks after implantation.
For identification of transgenic animals DNA is isolated from a small piece of the tail and tested for the presence of transgene by Southern blot PCR. To determine whether the transgene in cells of animal tgermline ransgenic mouse is crossed with another mouse. Further it is possible to conduct crossing descendants to obtain pure (homozygous) transgenic lines.

The described approach seems at first glance relatively simple, but it requires coordination of different stages. Even highly qualified specialist can obtain at best only 5% of viable transgenic animals from the inoculated eggs. None of the stages of the experiment is not effective at 100%, so for microinjection a large number of fertilized eggs should be used. For example, in obtaining transgenic mice after injection of DNA only 66% of fertilized eggs are survived; pups are developing from about 25% of implanted oocytes, and transgenic of them are only 25%. Thus, from 1000 implanted fertilized oocytes 30 to 50 transgenic mice are developing. In addition, introduced DNA can integrate anywhere in the genome, and often a lot of copies of it are included in a single site. And finally, not all transgenic pups will have the desired properties. In the body, some individuals can not express the transgene due to improper environment of integration site, and in the body of other foreign gene copy number may be too large, which can lead to overproduction of the protein and disruption of normal physiological processes. And yet, despite all this, the method of micro-injection is frequently used to produce lines of mice carrying functional transgenes.
Using of the modified embryonic stem cells. Cells from mouse embryos at the blastocyst stage, can proliferate in culture maintaining the ability to differentiate into all types of cells, including the cells of the germ line when they are administered to another embryo at the blastocyst stage. Such cells are called pluripotent embryonic stem cells (ES). ES-cells in culture easily modified by genetic engineering without compromising their pluripotency For example, at one nonessential gene site of their genome functional transgene can be integrated. Then it is possible to select the modified cells, cultivate them and use for the production of transgenic animals. This prevents accidental insertion characteristic by microinjection and retroviral vector systems.

During transfection of ES-cells in culture by vector aimed for the integration into a specific chromosomal site, in some cells, the DNA is embedded randomly, in other integration occurs at the right site, but in the majority ES-cell integration does not take place. To increase the number of cells of the first type so-called positive-negative selection are used. This strategy consists in a positive selection of cells carrying DNA vector which is integrated into the desired site, and negative selection of cells with DNA vector integrated in a random site.
Target site must be located in such a region of genomic DNA that do not encode critical proteins of development or cellular function. In addition, it is essential that integration of the transgene did not block translation of the corresponding region of the genome. Searching similar sites is conducted continuously.

A simpler way of identification of ES-cells carrying the transgene in the right site, is based on the of PCR. In this case, DNA vector contains two sections homologous to target site, one from the transgene and from the cloned bacterial or synthetic (unique) sequence, which is absent in the mouse genome. After transfection of ES-cells by this vector transfected cells are screened by PCR. One of the PCR primers complementary to the site of the cloned bacterial or synthetic (unique) nucleotide sequence of integrated vector, and the second PCR primers complementary to the site of chromosomal DNA adjacent to one of the homologous DNA regions.

When embedding of a target sequence in a random site the expected amplification product will not formed, and at the site-specific integration in the results of PCR amplification DNA fragment of known size is formed. Thus it is possible to identify pools ES-cells containing the transgene in the right site, and replanting cells of these pools it is possible to get the cell lines with the site-specific insertion.
ES-cells, in which the gene is integrated in the right site transgene, can be cultivated and put into an embryo at the blastocyst stage, and then implant such embryo into the uterus such pseudopregnant "surrogate" mothers. Pups in which genetically modified ES-cells involved in the formation of the germ-line cells, can give rise to transgenic lines. To do this, they should be crossed with individuals of the same line, and then cross their transgenic offspring. This will result in transgenic mice homozygous for the transgene.

Basically approaches to the creation of transgenic animals with "improved features" and the "loss of function" are similar. Unfortunately, pluripotent ES-cells similar to those in mice that are not found in cattle, sheep, pigs and chickens, but the search continues.
Transgenic mice can serve as model systems for the study of human disease and test systems to investigate the possibility of synthesis of products are of interest to medicine. Using whole animals emergence of disease as well as its development can be modeled. However, a mouse is not a man, but it also belongs to the class of mammals, and the data obtained from transgenic models can not always be extrapolated to man in terms of the medical aspects. However, in some cases, they can identify the key points of the etiology of complex diseases. Taking this into account, the researchers developed a "mouse" model of human genetic diseases such as Alzheimer's, arthritis, muscular dystrophy, tumors, hypertension, neurodegenerative disorders, endocrine dysfunction, cardiovascular disease, and many others.
To create transgenic cows a modified scheme of mice transgenosis by microinjection of DNA are used. The procedure includes the following stages: collection of oocytes of cows slaughtered at the slaughterhouse; oocytes maturation in vitro; fertilization of oocytes with bovine sperm in vitro; centrifugation of fertilized eggs to concentrate of yolks, which interferes with visualization of the male pronucleus in normal oocyte by sectional microscopy; microinjection of DNA into the male pronucleus; development of embryos in vitro; non-surgical implantation of a single embryo to recipient female during estrus; screening of descendants DNA for the presence of transgene.

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.

Genetic potential for growth in the populations of pigs, on the contrary, is not far from the potential plateau, and therefore the additional introduction growth hormone or transfer its gene does not give a significant effect in the speed of growth. Another explanation for the lack of accelerated growth of transgenic animals associated with the necessity of increasing not only the growth hormone, but some other, as yet unknown growth factors. Unregulated expression of the growth hormone gene, as autologous or heterologous, can lead to a reduction life expectancy of transgenic animals due to pathological disorders of metabolism, development of acromegaly (excessive growth of certain parts of the face, limbs and internal organs), and exposure to various infectious diseases. For example, diabetes - typical symptom of acromegaly was observed in transgenic sheep with high blood levels of growth hormone of cattle (S.E. Rexroad et al., 1990).

In another study, diabetes was observed in transgenic sheep, actively expressing their own growth hormone. Animals fell in the first year of postnatal life (CDNancarraw et al., 1991; KAWard et al., 1989). Transgenic pigs with hormone hypersecretion were lower on live weight at birth than littermates offspring, more sluggish, with a depressed appetite, susceptibility to arthritis, and most of them have not lived more than a year (VGPursel et al., 1987, 1989). It became apparent that a consistently high level of production of growth hormone in animals is not a positive factor in their productivity. Analysis of these experiments indicate that the use of transgenic technology to change the growth and composition of the tissue of domestic animals require further the understanding of the genetic regulation of growth. According to L.K.Ernst et al. (1993), directed influence on one hormone of growth-hormone complex cascade will not be effective until complex design with fine gene regulation of metabolic processes will not be created.

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 reached 35 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

Test questions: What methods are used to introduce of foreign DNA into animal embryos? 2. Gives examples of using transgenic animals for producing valuable products, 3. Tell us about the achievements of genetic engineering in breeding animals that are resistant to disease, 4. List the advantages of transgenic animals before the micro-producers of biological products 5. What recombinant proteins do you know which are derived from milk of farm animals? 6. Peculiarities of methods for creating transgenic birds and fish.

Lecture №9

Biotechnology of forages

A result of studying various organisms, it was found that many microorganisms are characterized by the high intensity of protein synthesis, and moreover microbial cell proteins have a high content of essential amino acids. Special experiments were conducted to determine toxicological evaluation of microbial protein, which indicate that the cells of some microorganisms can be used as concentrated feed additives, which are not inferior in the biological value of protein to soybean or fish meal.Microorganisms as a source of feed protein has several advantages in comparison with the plant, and even animals. They characterized by high (up to 60% dry matter) and stable content of proteins, whereas in plants the concentration of proteins varies considerably depending on the growing conditions, climate, weather, soil type, farming, etc. In microbial cells in addition to the proteins others nutritionally valuable substances: carbohydrates, lipids with a high content of unsaturated fatty acids, vitamins, macro-and micronutrients are formed. When using microorganisms on a limited area industrial production can be organized and produce a large amount of feed concentrates in any season, and besides microbial cells can synthesize proteins from waste of agriculture and industry, and thus, allow us simultaneously solve another major problem - the disposal of these wastes in order to protect the environment. Microorganisms have another valuable benefit - the ability to rapidly increase protein mass. For example, soybean plants of 500 kg in the maturation phase of seed can synthesize per day 40 kg of protein, and bull of the same mass - 0.5-1.5 kg, but yeast cells, weighing 500 kg - up to 1.5 tons of protein. Different types of yeast and bacteria, microscopic fungi, unicellular algae most often using as a source of feed protein.

Fodder yeast. Yeast are grown on hydrolysates of waste wood and other cellulose-containing plant material, which in the hydrolysis form carbohydrates easily assimilable for microorganisms. In the technology of obtaining feed protein residues cellulose and wood industry, straw, cotton hulls, sunflower baskets, flax, corn cobs, sugar beet molasses, potato pulp, grape squeeze, brewer's grain, little decomposed peat, bard of alcohol production, waste of confectionery and dairy industries are commonly used as a raw material. Chopped plant material containing a large amount of cellulose, hemicellulose, pentosans, are subjected to acid hydrolysis under increased pressure and temperature, in the results of 60-65% polysaccharides are hydrolyzed to monosaccharides. The resulting hydrolyzate was separated from the lignin. The excess of acid used for hydrolysis, is neutralized with milk of lime or ammonia water. After cooling and settling minerals, vitamins and other substances necessary for microorganisms are added to hydrolyzate. Obtained in this way growing medium is served in fermenter , where the cultivation of yeast is conducted. For cultivation on hydrolysates of vegetative waste are most effective yeast genera are Candida, Torulopsis, Saccharomyces, which can use as a carbon source hexose, pentose and organic acids. In optimal conditions, from 1 ton of waste softwood it is possible to get 200 kg of fodder yeast.
For obtaining of feeding yeast the deep cultivation technology in special apparatus – fermenters is used. Fermenters ensure constant mixing of microbial cells suspension in the liquid medium and the optimum conditions of aeration. Operating cycle of cultivation yeast culture takes about 20 hours. By the end of the cycle culture fluid with suspended yeast cells in it is derived from fermenter, and it is served again with nutrient substrate and culture of yeast cells to new cultivation. Suspension of microbial cells derived from the fermenter is fed to a flotation unit, with which yeast biomass is separated from the culture fluid. In the process of flotation foaming of suspension occurs. In this case microbial cells float to the surface together with the foam, which is separated from the liquid phase by decantation. After settling yeast mass is concentrated by the separator. For better digestibility of yeast in the body of animals special treatment of microbial cells (mechanical, ultrasonic, thermal, enzymatic) is carried out, providing the destruction of their cell membranes. Then yeast mass is evaporated to the desired concentration and dried (moisture of the finished product should not exceed 8-10%). The dry yeast mass contains 40-60% crude protein, 25-30% of digestible carbohydrates, 3.5% crude fat, 6.7% fiber, and mineral elements, many vitamins (up to 50 mg%). By treating yeast with ultraviolet rays it is possible to enrich it with vitamin D2, which is formed from ergosterol contained in them. To improve the physical properties of the final product feed yeast is produced in granular form.
In Russia and other oil-producing countries have developed technology for production of fodder yeast from n-paraffin of oil. Yeast cells can use as sources of carbon straight-chain hydrocarbons with carbon numbers from ten to thirty. They are liquid fraction with boiling temperature of 200-320 ° C, which are isolated from its oil by distillation. Candida guilliermondii is the most effective strains of yeast for cultivation on n-paraffin oil. Isolation and drying of yeast mass is conducted approximately by the same technology as in the hydrolysis production. Dried yeast mass is granulated and used as a protein-vitamin concentrate (PVC) for animal feeding, containing up to 50-60% of proteins.
National Center for Biotechnology of the Republic of Kazakhstan developed the new technology for obtaining of microbiological protein feed for animals. Several versions of feed additives are obtained: feed yeast based on Saccharomyces cerevise, Candida tropicalis CK-4, as well as microbial L-lysine on the basis of lysine-producer Brevibacterium 92. Experiments were carried out on submerged cultivation Candida tropicalis CK-4 in fermenter using as a carbon substrate glucose in fed-batch regime. Conditions of cultivation of lysine producer in batch-mode are developed. Experimental batch of animal feed additive based on the yeast Saccharomyces cerevise (S.K.Barbasova et al., 2011) is obtained.
Protein concentrates from bacteria. Along with getting fodder yeast bacterial proteins containing crude protein content of 60-80% by dry weight are also important for forage. There are more than 30 species of bacteria, which can be used as a valuable source of feed protein. Bacteria are able to increase biomass several times faster than yeast cells and in the protein of bacteria contains much more sulfur-containing amino acids owing to what it has a higher biological value than the protein of yeast. Various gaseous products (natural and associated gas, gas condensate, etc.), lower alcohols (methanol and ethanol), hydrogen are serving as a carbon source for the bacteria. Methylococcus most often are grown on gas nutrient media and under optimal conditions can utilize up to 85-90% methane. Due to the fact that the gas atmosphere from methane and air is explosive and better utilization of methane by bacteria requires constant recycling, production of feed protein from the gaseous products is quite complex and expensive. Technology for growing bacterial protein mass on methanol, which can be easily obtained by the oxidation of methane is widely used nowadays. During cultivation in a nutrient medium containing methanol, bacteria from genera Methylomonas, Pseudomonas, Methylophillus are most effective. These bacteria are grown in normal fermenter using a liquid growth medium.
Large-scale production of food proteins based on the use of methanol was first organized in England. Concern «ICI» produced forage protein preparation with the trade name "Prutin." Russia also has the technology for production of bacterial protein mass from methanol (commercial name of preparation is "Meprin."). It contains in its composition up to 70-74% of proteins, 5% lipids, 10% minerals, 10-13% of nucleic acids. On the basis of cultivation Acinetobacter technology of getting feed protein from ethanol (preparation name is "Eprin", which may have also edible) is developed.
Hydrogen-acidified bacteria are characterized by high intensity of synthesis of proteins that can accumulate in their cells up to 80% crude protein of dry matter. These bacteria use the energy of the oxidation of hydrogen for carbon dioxide utilization, and some strains also for the assimilation of atmospheric nitrogen. For the cultivation of bacteria in the hydrogen-gas atmosphere usually contains 70-80% hydrogen, 20-30% oxygen and 3-5% carbon dioxide. High efficiency of growing in such gaseous medium have bacteria of the genera Pseudomonas, Alcaligenes, Achromobacter, Corinebacterium etc.

Hydrogen for the production of protein mass is usually obtained from water by it electrolysis (electrolysis) or photochemical decomposition. Carbon dioxide can be used from any industrial waste gas industries, as well as flue gas, which simultaneously solves the problem of cleaning the gas medium. Production of feed protein on the basis of hydrogen-oxidizing bacteria can also be arranged near the chemical plants, where by-product hydrogen is formed.

Feed protein from algae. In Russia and other countries for the production of feed protein unicellular algae Chlorella and Scenedesmus, as well as blue-green algae of the genus Spirulina are used, which are able to synthesize proteins and other organic substances from carbon dioxide, water and minerals by the assimilation of the sunlight. For their growing it is necessary to provide some light and temperature regimes, as well as the large volume of water. Under natural conditions algae are grown in the southern regions using the open pools, but the technologies of their cultivation in a closed system are also being developed.
Chlorella and Scenedesmus require for their growth neutral environment, their cells have a rather dense cellulose casing, so that they are badly digested in the body of animals. Destruction is held by special treatment to better digestibility of cellulose casing.

Spirulina cells 100 times bigger than chlorella, but they have no sound cellulose casing so it is best digested in body of animals. Spirulina is grown in alkaline (pH 10-11), under natural conditions in alkaline lakes.
By the intensity of biomass accumulation algae considerably exceed agricultural plants. At growing it in open cultivators it is possible to get up to 70 tons of dry biomass per year from 1 ha of water surface, whereas in growing wheat - 3-4 tons, rice - 5 tons, soybeans - 6 tons, corn - 7 tons.

Technology for producing protein mass from algal cells includes growing industrial culture in the open or closed cultivator, separation of algae from water mass, preparation commercial product in the form of suspensions, dry powder or paste-like mass. The process of separation of algae cells from the mass of water is the energy consuming because it is necessary to process large volumes of liquid.
In Russia cultivation of chlorella is most widespread, which is used for animal feeding in the form of a suspension (1.5 g / l of dry matter) or dry powder. Daily rate of Chlorella suspension for feeding young cattle is equal to 3-6 L, adult animals - 8-10 liters. It is important to grow algae on the wastewater industry, thermal power plants, livestock facilities, as in these cases, along with obtaining feed protein simultaneously solve the problems related to the protection of the environment. For example, the cultivation of Scenedesmus or Chlorella at sinks of livestock complexes for 15 days allows to completely clean it from organic substances, and the smell and color are disappeared. During cultivation of algae in industrial wastewater or in wastewater of thermal stations excess heat rejected from these objects is used and carbon dioxide which is formed as a by-product of the process and the burning of various wastes is utilized.

There are of open type of cultivators for growing algae in many countries. The largest firms in growing Chlorella "Chlorella Sun Company" is situated in Japan. In Bulgaria, on the waters of thermal springs Chlorella and Scenedesmus are cultivated, and Bulgarian scientists have managed to obtain strains of Chlorella without cellulose casing, so that the biomass of these cells is well digested in the body of animals. In a significant number of protein concentrates from Spirulina are produced in central Africa and Mexico, where there are alkaline lakes. The largest manufacturer of a variety of products from biomass and Spirulina protein is the company «Coca Texcoco" (Mexico). In Italy, technology for growing cells Spirulina on sea water and closed cultivators are being developed. Due to the fact that biomass of algae of Spirulina is easily digested with gastric juice and has a high protein content (70% dry matter) well-balanced amino acid composition, in some countries it is used for cooking food, especially pastry, enriched the protein.

Considering the importance of introduced to the industrial culture of algae as an additional source of complete protein for animal feeding and nutrition, Research to improve existing industrial strains of unicellular algae, and getting new genotypes that should combine a high rate of photosynthesis, cold resistance, good digestibility, the ability to synthesize large amounts of protein quality (high content of essential amino acids), and better utilize the substrate is conducting by scientists from different directions - breeders, geneticists, biochemists. Important role in the implementation of such studies is given to genetic engineering.

Proteins microscopic fungi. Valuable source of well-balanced amino acid composition of proteins are the mycelium cells of many microscopic fungus. For its nutritional properties fungi proteins approaching soy protein and meat, so that can be used not only for the preparation of feed concentrates, but also as an additive in human food. The raw material for commercial cultivation of microscopic fungi are usually the vegetable wastes containing cellulose, hemicellulose, lignin. At the same time two critical issues are solving: getting protein mass and recycling plant, woodworking and paper industry, which can be a source of environmental pollution. Particularly important to find active strains of microorganisms that can utilize carbon of lignin, which is highly resistant to degradation by microflora. In nature, lignin is decomposed only by brown and white rot fungi of Stropharia, Pleurotus, Aborkporus, Coriolus, Stereum and others. Currently in the process of studies, the fast-growing non-toxic strains of the meso-and thermophilic fungi from the genera Penicilium, Aspergillus, Fusarium, Trichoderma for industrial cultivation is selected. Mycelium cells of these fungi have a thin cell wall owing to what it is a very well-digested in the gastrointestinal tract of animals. They contain in their composition complex of aromatic substances rich in vitamins and easily digestible lipids.

As compared with yeast proteins microscopic fungi contain high amounts of sulfur amino acids and fungi proteins have the better digestibility. Concentration of nucleic acid in fungi mycelium (1-4% of dry weight) is almost the same as in the tissues of the plant. At the same time in the biomass of fungi protein is much less than in yeast (20-60% of dry weight) and fungi grow more slowly (doubling biomass in 4-16 hours, whereas in yeast 2-3 hours). Certain microorganisms are of great interest as probiotics. Probiotics are biological products containing live microorganisms - symbionts of humans and animals that have the ability to restore the disturbed Microecology of organisms. These include bifidobacteria, lactobacilli, streptococci, etc., present in the body at birth. In veterinary practice for the development of probiotics, besides the genera of bacteria, yeast and fungi (Saccharomyces cerevisiae, Candida pintolonesi, Aspergillus niger, Asp.oryzae) are also used.

The raw materials used for the preparation of culture media, must be harmless to humans and animals. Main substrate is skimmed milk and hydrolysed milk. The biomass of microbial symbionts is concentrated by separators, further components of supporting medium during storage are introduced (gelatin, sugar, skim milk), then it filled into ampoules or vials and aliquots are lyophilized. In the technologies of creation of probiotics lactic acid bacteria, bacilli less frequently, Escherichia and others are mainly used. For example, Baktolakt (Japan) includes Lactobacillus rhamnosum, Lactobacillus casei, Lactobacillus faecium: Linex (Russia) consist of Lactobacillus, Bifidobacterium, Streptococcus; Biosoprina (Ukraine) has in its content Bacillus sibtilis, Bacillus cereus.

In biotechnology are important not only the microorganisms, but also their metabolites, namely amino acids, vitamins (primary metabolites), antibiotics, etc.
Amino acids. In the world 700-800 thousand tons of amino acids (more than half of glutamine and lysine) are produced annually. There are about 300 different amino acids. In living nature 20 amino acids are used and 8 of which (izoleitsin, leucine, lysine, methionine, threonine, valine, phenylalanine, tryptophan) are essential for human. Last enter the body with the protein of animal and vegetable origin. Amino acid composition, their quantity in cells of the animal, plant and microbial origin have some differences. Thus, in the proteins of plants lysine, methionine, tryptophan, threonine are not sufficient. Most organisms, except lactic acid bacteria and some other groups, in contrast to higher organisms are able to synthesize all 20 amino acids.

Commercially protein amino acids are obtained: by hydrolysis of natural protein-containing raw materials; chemical synthesis; microbial synthesis; biotransformation of precursors of amino acids using microorganisms or enzymes isolated from them. Microbial synthesis of amino acids is the most promising and economically profitable. Industrial production of amino acids became possible after the discovery of the ability of some microorganisms secrete into the culture medium significant amounts of certain amino acids. There have been no any connection between taxonomic position and the ability of micro-organisms to the production of some amino acids. Thus, among the possible glutamic acid producing organisms 30% are yeast, 30% - streptomycetes, 20% - and 10% of bacteria (microscopic fungi). Only one of the examined strains of microorganisms - Corynebacterium glutamicum was capable for overproduction of glutamate. This strain was used in the organization of the world's first large-scale production of glutamic acid by microbiological method in Tokyo (1956). This amino acid has been widely used in the food industry to improve the taste.

Prospective producers are constantly improving by means of selection of mutants with an altered genetic program and regulatory properties. To them, except Corynebacterium, can be referred strains of microorganisms such as Brevibacterium, Micrococcus, Arthrobacter, etc.
Carbon source for producing strains Corynebacterium glutamicum are glucose, sucrose, less fructose, maltose (in the molasses, whey, casein hydrolyzate, etc.). The nitrogen source may be urea, ammonium sulfate or phosphate, corn steep liquor, yeast. As growth promoters corn extract, yeast hydrolyzate, vitamins of group B, macro-and micronutrients (Ca, Mg, Mn, Fe, P) are used.

Lysine is synthesized on an industrial scale, primarily as a feed additive. In practice lysine is obtained by deep-batch fermentation of Brevibacterium flavum and Corynebacterium glutamicum at 30-33 ° C, pH 7.0-7.2 within 2-3 days. Lysine is accumulated in culture medium at the end of the exponential phase of growth. At the end of the fermentation culture liquid is separated from the cell mass. Lysine is separated from the culture fluid, mixed with filler (wheat bran, etc.), and used as feed concentrate in granulated or liquid form. Granulated lysine contains 7-10% of lysine. In the cells of microorganisms lysine is synthesized from aspartic acid and serves as the end product of a branched biosynthetic pathway common to three amino acids - lysine, methionine and threonine.

Vitamins. Biological activity of vitamins is determined by what that they are part of the active sites of enzymes as cofactors. Therefore, the lack of vitamins decreases biocatalytic activity of enzymes, affects metabolism, growth and development of organisms. Biosynthesis of vitamins in natural conditions carried out by plants and microorganisms. In the processing of plant food loss of vitamins is often observed. So in obtaining upper class flour 80-90% of the vitamins is lost.
As bio producers are used single-celled microorganisms, actinomycetes, methane-producing, photosynthetic bacteria, including more than 10 species of propionic acid bacteria. Propionibacterium ari is selected, which is capable for active secretion of B12 from cells, in contrast to other producers of this kind, which accumulate vitamin within the cell. The product is produced by submerged cultivation of producing strains under anaerobic conditions on a substrate containing corn extract, glucose, salts of cobalt and ammonium sulphate. Riboflavin (vitamin B2) is synthesized by higher plants, yeast, filamentous fungi and bacteria. From 1 ton of carrots 1 g of riboflavin can be obtained, and from 1 ton of liver - 6 g, whereas during cultivation of industrial strains - Eremothecium ashbyii or Ashbya gossipii in1 tonne of nutrient medium 25 kg vitamin is accumulated. Mutant strains of B.subtillis Asp.niger are also used as producers. Cultivation of strains is carried out in fermenters with constant aeration. As a substratum soy flour, molasses, whey, fish and corn meal are used. Saccharomyces carlsbergensis and S.cerevisiae ) are used as producers of ergosterol (a precursor of vitamin D2 – calciferol). Yeast fermentation is carried out in conditions of aeration. The resulting biomass is hydrolyzed with hydrochloric acid and then cleaned with alcohol, concentrated and irradiated by UFL with wavelength of 280-300 nm. Radiation excites certain chemical bonds in carbon cycle causing conversion of ergosterol to vitamin.

Large-scale production of another vitamin - L-ascorbic acid (vitamin C), which is not less important to the human body and animals, is a time-consuming process that involves a number of microbiological and chemical stage. Initial substrate for it is D-glucose. In the last stage of the process 2-keto-L-gulonic acid (2 - KLG) is converted chemically to L-ascorbic acid.
Biochemical studies of the metabolism of various microorganisms have shown that 2 - KLG can be obtained by co-culturing Corynebacterium and Erwinica herbicola for the conversion of glucose to 2 - KLG. However, the culture conditions which are optimal for one microorganism, are not acceptable for the other, which leads to spontaneous "washout" of them from the culture medium. In such cases, microorganisms can be cultured sequentially, but such process is difficult to make a continuous since for the growth of micro-organisms are very different environment are necessary.

The easiest way for creation a single microorganism capable of converting D-glucose to 2 - KLG is to separate the gene 2 - KLG-reductase of Corynebacterium and inserting it into Erwinica herbicola.
Transformed cells of Erwinica herbicola actively convert D-glucose directly into 2 - KLG. In this case, enzymes of Erwinica herbicola localized in the inner membrane of the bacterial cell, convert glucose to 2,5-DKG (2,5-diketo gluconic acid) and 2,5-DKG-reductase, localized in the cytoplasm, catalyzes the process of turning 2.5 - DKG to 2 - KLG. Therefore, by means of genetic manipulation it is possible to implement metabolic reactions proceeding in different microorganisms in one organism. This hybrid has acquired the ability to synthesize the final product of the combined metabolic pathway. Such an organism is used as a factory for the production of 2 - KLG, replacing the three stages of the process of producing L-ascorbic acid, which dominates today.

Antibiotics like pigments and toxins belong to secondary metabolites of microorganisms, ie substance is not a essential for the growth and functioning of cells, but are synthesizing in the stationary phase. They are used in animal husbandry, not only as a medicine, but as a feed additive. With the properties of promote animal growth have more than 20 antibiotics which are synthesized by filamentous fungi (biomitsin and terramycin) and streptomycin (Grisinum, flavomitsine, monensine, tylosine). As filling of the feed additive is commonly used soy flour.
Biosynthesis of antibiotics, as well as any other secondary metabolites, increases in the phase of slower growth of the cell population) and reaches a maximum in the stationary phase (idiophase). It is believed that at the end of trofophase enzymatic status of cells is changes and inducers of secondary metabolism are appeared, releasing secondary metabolism genes from the influence of catabolite repression. Therefore, any mechanism, inhibiting cell proliferation and active growth or stress activate the formation of antibiotics.
Idiolit cultivation process passes through two phases. In the first phase accumulation of a sufficient amount of biomass, which is cultivated on a medium for microbial growth is occured. This phase should be fast and culture medium should be cheap. In the second phase synthesis of the antibiotic is launched. In this phase fermentation is conducted on productive medium.

Test questions: 1.List raw materials and genera of yeast which are used to produce feed protein 2. What are the sources of carbon and species of bacteria used in the production of protein concentrates; 3. What is the technology of producing protein mass out of algae cells? 4. Tell us about the modern production of probiotics, amino acids, vitamins and feed antibiotics

Lecture №10
Biotechnology and biosafety

Integration donor heterologous gene in recipient cell DNA is associated with certain difficulties. The most important of which is ensuring targeted insertion of a gene or group of genes, as well as its normal function, ie expression. This problem is always present and its solution in many cases still has a random nature.Even more important is the problem of the genetic risk, the possibility of getting mutants containing toxic or allergenic proteins for human or other dangerous compounds. The real risk with the behavior of a foreign gene into a recipient cell, hypothetically always exists. First of all, it can be caused by pleiotropic effects during interaction and interchangeability of genes. According to KG Ghazaryan, destabilization of the genome during transgenosis can occur not only due to the enrichment of the genome with new genes or mutagenic effect of insertion but possibly due to the induction of endogenous systems of recombination and activation of "silent" genes. All this give reason to believe the possibility of appearance dangerous genotypes to human health in transgenosis. The risk of getting these mutants significantly increases when using of artificial, synthetic genes for the production of transgenic plants, animals and microorganisms with improved and radically new properties. These circumstances justified concern of many people, their insistent demand to prohibit creation and especially using of genetically modified organisms and derived from them food and other products.
For these two reasons can be added a third one - the spontaneous transfer with pollen of modifier genes in other plants and their interaction with genes of third genotype, which could lead to the emergence of new genotypes with dangerous properties to humans and the environment.

It is known that the beginning of the debate on the issue of biosafety in science and society put the scientists - the founders of a new direction - bioengineering. In 1974, eleven of the world's leading molecular biologists led by the father of American Genetic Engineering P. Berg, who created the first recombinant DNA molecule, appealed to international community with a letter through the journal «Science», in which they proposed to abandon experiments with recombinant DNA before the International conference on this issue. However, already in 1975, at a conference in Asilomare (USA) scientists have concluded that experiments in genetic engineering, the latest biotechnology, no more dangerous than similar work in other industries, but it is necessary strict control over biosecurity measures. In 1976, in the USA the first regulations handling of recombinant microorganisms were taken. Releasing them outside the walls of laboratories were forbidden. At the end of the 70's in most countries worldwide legislation was appropriated. Gradually, these rules were corrected towards mitigation of severity. Thirty years of intensive work in the world in the sphere of modern biotechnology (genetic engineering) have confirmed their safety.

In the laboratories performing genetic engineering research and obtaining of transgenic organisms, which are not related to biological weapons of humans and nature, no cases of obtaining genotypes of plants and animals dangerous for human health and life, as well as the environment is not registered. Microbiologists are working purposely to strengthen or weaken the virulent and other properties of bacteria, solving a number of problems of medical biosafety and protection of states from biological weapons and aggression. Unfortunately, the world terrorism does not stop before selecting resources for their crimes. It uses for this purpose life-threatening biological resources. For world community it is necessary to urgently develop and implement a system of the most effective measures to combat terrorism and to prevent the use of achievements of life sciences in his sinister purposes.

Scientists are able to provide long-term stability of the Biosafety in bioengineering. It can be explained by the following main points. First, the bioengineers used in their works of natural genes that during the whole evolution participated with recombinogenesis, undergo selection and elimination owing to what have developed mechanisms at all levels of biological objects that ensure sustainability repair of biosynthesis of proteins and their quality. Second, in all bioengineering laboratories effective methods of monitoring the quality of transgenic organisms (properties of proteins and other components of the newly created genotypes) are developed. This allows in advance at the creation stage of genetically modified organisms (GMOs) in the laboratory, to identify genotypes hazardous to human and environmental and prevent their release from the laboratory for use in production. According to most genetic engineers, systematic monitoring equipment for the development and use of GMOs needs to be improved. New techniques for early detection of toxic and allergenic substances in transgenic objects covering groups and classes of low molecular nature should be also developed. And third for creating genetically modified organisms experts select well known and proven natural regulatory genes and their genetic structure. Vectors created on their basis provide the obtaining transgenic with given properties. Finally it provides creation of new genotypes safety for people and the environment. In general, the situation with genetic engineering research on transgenosis should remain under the strict control of the scientists and the government. According to some researchers the technology for producing transgenic animals is far from perfect. The unpredictability of results carrying over foreign genes and the presence of unexpected effects limit practical application of transgenosis. Researches of bioengineering centers should actively develop investigations on improving the technology, methods, techniques and criteria for biosafety of GMOs. It is only on this basis that they will be able to accelerate the development of fundamentally new genotypes of plants, animals and microorganisms to improve the sustainability and productivity of agricultural production, solving complex problems of modern medicine, and other fields of science and economics.
Criteria, indicators and methods for the assessment of genetically modified organisms and products derived from them on biosafety. An important stage of biosafety assessment of genetically modified organisms and derived food and other products are sanitary and hygienic expertise. And there following criteria should be checked: the chemical composition of the original and transgenic plants, whether it is worth the biological value and digestibility of products made from GMO; whether GMOs and products derived from it causing allergies or affect the immune system; will they prove toxic, carcinogenic or mutagenic; may be foreign genes affecting to the reproductive functions of animals and humans; whether introduced gene will be transferred into other organisms and will be transmitted to descendants of plants; whether a new gene affecting plant resistance to diseases; do the transgenic plants affect on soil microflora and other parts of the biocenosis, etc. Biomedical evaluation of food products derived from GMOs is mandatory and also extremely important. For example, Russia has developed methodical instructions "Medical and biological evaluation of food products from genetically modified sources." Methodological guidelines are installed order for hygienic examination and registration of food products derived from genetically modified sources. Methods of medical and hygienic, medical and biological evaluation and clinical testing of new types of food products derived from genetically modified sources are approved. Methodological guidelines are official publications and their implementation are strictly controlled by Ministry of Health of the Russian Federation, as well as relevant legal and regulatory bodies

Peculiarities of the state regulation of genetic engineering and biosafety controls production and use of GMOs in the U.S.. States ranks first in the world in production of genetically modified products. There are laws and regulations of Congress and President of USA permitting the use of GMOs in production. A half of soybean and quarter of the corn crops in private farms are occupied by transgenic varieties and hybrids. For official registration genetically modified organisms three agencies are responsible - Ministry of Health, Ministry of Agriculture and Ministry of Environment.Each of these ministries make their own independently decision on registration or rejection from registration of modified organisms,. A positive decision can be taken only with the consent of all three agencies. Service of veterinary inspection and plant protection of the Department of Agriculture (USDA) in accordance with established rules and procedures of notification (notification) takes decisions about the interstate movement genetically modified organisms, on import and release them into the environment. These rules were first published and came into force in March 1993.

The timely establishment of an effective legal framework of biotechnology and bioengineering has been another factor providing accelerated development of science and practice in the field of genetic engineering. The main direction in bioengineering is the creation of genetically modified varieties and hybrids of soybeans, corn, cotton, sugar beet, potatoes, tomatoes, canola and other crops that are resistant to the total herbicide Roundup (glyphosate), fungal diseases and insects. Intensive research is underway to create varieties of wheat and other crops that are resistant to fungal and viral diseases. The greatest success in creating resistant varieties and hybrids of the above crops have achieved by researchers of world-famous company "Monsanto" (St. Louis, MO).

Farmers do not have and do not put forward any problems with the cultivation and sale of seeds and grains of genetically modified varieties and hybrids of soybeans, corn and other crops. Due to modified plants they receive substantial addition to the profit by reducing the cost of herbicides and pesticides and care for crops. Mandatory labeling of food derived from genetically modified varieties and hybrids has not yet entered in the United States. But at the request of customers it can be introduced at any trading enterprises at any time.

Kazakhstan – signatory of Cartagena Protocol. Surveys on regulation of GMOs based on Cartagena Protocol of Biosafety, allowed Kazakhstan to develop projects for "Concept of state control and regulation of GMOs in the Republic of Kazakhstan", and a number of regulations, and create the conditions for the formation of the system of regulation in the Republic of Kazakhstan. The analysis showed that practically no country has legislation that can prevent unpredictable consequences in the creation, use and dissemination of GMOs. Today there are two different principles in solving problems of regulation of GMOs: "precautionary", which adheres to the EU and the principle of "substantial equivalence", which adheres to the U.S., Australia, Canada and other countries E.M.Ramankulov, 2011). The monitoring showed that food products containing GM ingredients are entering to the markets of RK without control. It is advisable for Kazakhstan to stick in its policy of the precautionary principle, which is based on the extreme caution in the use of GMOs, and requires the introduction of labeling "product contains GMO", which is very important to ensure food safety. Important provisions of the Cartagena Protocol is the right to carry out assessment of the risks of the genetically engineered products for a decision on their import. As part of the assumed obligations, the Government of Kazakhstan has designated the Ministry of Agriculture as National Focal Point, and Ministry of Education and Science as the competent authority. Wherein the first ministry is the point of contact between Kazakhstan and the Secretariat of the Protocol, and the second is responsible for the administration. National Center for Biotechnology is defined as the center of the Republic of Kazakhstan for implementation of a Biosafety Clearing-House (BCH). The latter acts as the central market of information, on which the mutual exchange of operational information between all parties Biosafety Protocol is occured. Currently, a project of Law "On state regulation of genetic engineering" is developed, which is currently under consideration by Parliament. The objectives of this bill are the protection of public health, protection of the environment during use of GMOs, biological diversity, ensuring the security of the country in the implementation of genetic engineering, the development of genetic engineering, etc.

The reaction of the international community on the accelerated development of biotechnology and bioengineering in the leading countries of the world. In many EU countries public attitudes towards the development of biotechnology is negative, mainly to the creation and use of recombinant-modified organisms. The European Parliament and the government of EEC took a number of special documents, limiting and prohibiting further release into the environment of genetically modified plants and other organisms At the same time, the U.S., UK, France, Eastern Europe, adopted important government decisions in support of the Biotechnology and Bioengineering, authorizing the use of genetically modified varieties and hybrids of crops. There are no scientists among the active opponents of bioengineered modifications , most of them are politicians, businessmen and media representatives. Science-based, proven arguments against the development and use of GMOs and derived products did not raise by them. They give facts which turned out to have no relation to transgenic organisms. Science-based prognosis of events around the issue of transgenic organisms testifies that the public protests in the world, including in Russia, has already reached its peak and in the case of strict compliance with all legal requirements and in-depth scientific monitoring of bioengineering it will gradually subside in the future. Countries that artificially put forward various reasons that delay the development of biotechnology and bioengineering, and the use of their achievements in the production ultimately will suffer significant economic damage, because the volume of the most important biotechnological products in the global market will continue to grow, and they will have to spend a large part of their currency resources on the purchase of these products.

Test questions: What are the criteria, indicators and methods for the assessment of genetically modified organisms and products derived from them? What measures are carried out in the Republic Kazakhstan in the framework Cartagena Protocol?

Дата добавления: 2018-04-15; просмотров: 418; Мы поможем в написании вашей работы!

Поделиться с друзьями:

⇐ Предыдущая 1 2 3 4 56

Мы поможем в написании ваших работ!