Features of the methodology for creating transgenic birds and fish
Transgenic birds. Microinjection of DNA into fertilized egg cells of birds in order to obtain transgenic lines is not an easy procedure. This is due to some features of the reproduction and development of birds. Thus, when fertilized in birds, several spermatozoa can enter the egg at once, and not just one, as is usually the case in mammals, and it is impossible to identify that male pronucleus that connects with the female one. The microinjection of DNA into the cytoplasm is also not suitable, because in this case DNA does not integrate into the genome of a fertilized egg. Finally, even if micro-injection of DNA into the nucleus can be carried out, further operations will be difficult to implement, since in birds the egg after fertilization is quickly enveloped by a strong membrane, covered with a layer of albumin and inner and outer lime shells.
However, the transgene can be introduced into the yolk region (embryonic disc), which contains both female and male pronuclei and is formed earlier than the shell. After the introduction of DNA, each egg is cultured in vitro, and when the embryo is formed, it is placed in a surrogate egg to mimic hatching. With this strategy, one line of transgenic chickens was obtained. However, at present this method is inefficient and technically difficult to perform under normal conditions.
By the time the outer lime shell of the ovum of birds has hardened, the embryo at the blastoderm stage consists of two layers of 40,000 and 80,000 cells. Experiments have been conducted on inoculating such an embryo with retroviral vectors with altered replication carrying bacterial marker genes. As a result, transgenic chickens and common quail, carrying foreign genes in the cells of the germ line, were obtained. Typically, such birds do not produce free virus particles and, nevertheless, the use of retroviral vectors as "suppliers" of alien genes to animals that can then be used for food inevitably raises questions about the safety of such an approach. In addition, the size of the transgene that can be introduced into the recipient organism as part of the retroviral vector does not exceed 8 tons of DNA, and in some cases the integration into the source site is unstable. All this made the researchers look for alternative ways of transgenesis.
No bird-specific ES cells were detected, so the approach based on their use is not applicable to birds. More promising is a method using recombinant embryonic cells. It consists in the following. Blastoderm cells from the chick embryo are isolated, transfected with cationic lipids (liposomes) associated with transgenic DNA (liposomal transfection), and reintroduced into the sub-embryonic region of freshly-laid eggs. Some of the descendants will carry in a small amount of donor cells: these animals are called chimeras. In some chimeras, cells derived from transfected cells can form germline lines, and after several rounds of crossing such chimeras, lines of transgenic animals can be obtained. To increase the chances of creating chimeras carrying foreign genes in germ cell lines, the number of donor cells in chimeras can be increased by irradiating the recipient embryos before transfected cells are introduced into them. Under the influence of irradiation, some (but not all) blastoderm cells will die, and the ratio between transfected cells and recipient cells will increase in favor of the former. Apparently, in this way it is possible to obtain transgenic chicks, although with low efficiency.
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Transgenic chickens can be used to improve the genotypes of already existing breeds - to give them (in vivo) resistance to viral infections and diseases caused by coccidia, to increase the efficiency of digestion, to lower fat and cholesterol in eggs, and to improve the quality of meat. It has also been proposed to use an egg with its high protein content as a source of protein products used in the pharmaceutical industry. Expression of the transgene in the cells of the chicken's reproductive pathway, where a large amount of ovalbumin is usually secreted, can promote the accumulation of the corresponding protein product in the egg, from which it can then be isolated.
Transgenic fish. With the depletion of natural fish stocks, an increasing role will be played by fish farming under artificial conditions. The main goal of research in this area is the creation of recombinant fishes by transgenesis. Until now, transgenes have been injected with microinjection of DNA or by the electroporation of fertilized eggs of various fish species - carp, catfish, trout, salmon, etc. Since in fish the pronucleus in a fertilized egg is poorly discernible in a conventional microscope, linearized transgenic DNA is introduced into the cytoplasm of fertilized eggs or cells embryos that have reached the stage of four blastomeres. Embryogenesis in fish flows in an aquatic environment outside the body, so implantation is not necessary. All further processes can take place in tanks with a controlled temperature. Survival of fish embryos after microinjection is rather high, from 35 to 80%, and the proportion of transgenic offspring varies from 10 to 70%. The transgene can be detected by PCR using either embryonic red blood cell preparations or total DNA. Crossing transgenic fish, you can derive transgenic lines.
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Most of the first studies in this area were aimed at studying the effect of growth hormone transgene on growth rate. In one experiment, a transgene was introduced into the ovum of Atlantic salmon, consisting of the following elements: the promoter of the gene for the antifreeze protein of the American tubercle, the salmon growth hormone cDNA, the termination / polyadenylation signals of the 3'-end of the antifrice protein gene of the American tubercle. As a rule, transgenic salmon were larger and faster in weight than control untransformed individuals. In this case, an expression system was chosen with accelerated transcription of the growth hormone gene in cold water and suitable for "all fish", which avoided the biological incompatibility that might occur if the growth hormone gene did not originate from fish. The one-year-old transgenic specimens obtained as a result of the introduction into the ovum of the genetic construct of growth hormone suitable for "all salmon" weighed about 11 times more than the non-transgenic ones. The physiological activity of the lines of such transgenic salmon under natural conditions is of considerable interest. It is assumed that in the future, resistance genes to diseases and stresses, as well as genes that cause other biological characteristics, will be introduced both to fish of temperate latitudes and tropical fish.
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