Obtaining animals by homozygous diploid offspring and cloning technology



Obtaining homozygous diploid offspring. In purebred breeding, linear breeding is traditionally used, the purpose of which is to maintain a high genetic similarity with an outstanding ancestor. This is achieved through moderate inbreeding and targeted selection. Animals of such lines, having a high degree of homozygosity, are distinguished by a great genetic similarity. In this case, a high phenotypic homogeneity is created within the line in relation to physiological and morphological features. Unfortunately, the creation of inbred animals takes a long time, since it is associated with the splitting and recombination of genes, low fecundity and a large interval between generations.

The method of obtaining homozygous diploid offspring is similar in many respects to the technology of nuclear transplantation from somatic cells into an enucleated zygote. However, in the latter case, heterozygous animals are obtained, and not homozygous for all the genes of one of the parents. The essence of the method is as follows. From a zygote located at the stage of two pronuclei, the male or female pronucleus is microsurgically removed, leaving only one haploid set of chromosomes - male or female. To develop a zygote containing a haploid set of chromosomes, its activation or restoration of a diploid set is required. For this purpose, a short-term incubation of the haploid zygote in a solution with cytochalasin B is carried out. The latter interferes with the first cell division, but activates the fission of the nucleus and diploidization of the remaining pronucleus. Once the fission of the nucleus has occurred, the embryo is washed away from it, so that the number of chromosome sets does not increase. Experiments with transplants of pronuclei between the zygotes of the mouse showed that mammals require both male and female pronuclei for the normal development of the embryo, up to the time of birth. It is believed that a diploid genome, obtained only from one parent of the same sex, can not ensure the normal development of the embryo. It is believed that the father's genome is required for the formation of extraembryonic tissues, and the mother's for the passage of certain stages of embryogenesis. The new data need theoretical understanding and experimental confirmation. There are deep and long-term studies to overcome the methodological difficulties in obtaining homozygous diploid animals.

Cloning animals is obtaining identical descendants by transplanting the nuclei of embryonic cells into sex cells with removed nuclei. The production of five 32-cell blastomer from a high-yielding donor cow and the transplantation of each nucleus into an enucleated egg allows 160 embryos from the donor simultaneously. When this procedure is repeated with the received "secondary" embryos, the possibilities for obtaining an unlimited number of offspring are opened.

For the first time, they transplanted the nuclei on the amphibians Briggs and King in 1952. They showed that the nuclei from early embryonic cells retain the ability to further develop after transplanting them into enucleated eggs. Kernels are specialized, i.e. the somatic cells of adult frogs lost totipotency (the property of cells to fully realize their development potential with the formation of the whole organism), and at best the development of genetic copies occurred up to the early stages of tadpoles. They also found that nuclei from embryos at a later stage of development, as well as from less developed embryos when transplanted into an egg, do not break up. Newport and Korscher (1982) explain this by the differentiation of embryonic cells, which manifests itself in the midblastula rearrangement stage, i.e. at the stage at which the embryo begins to produce its own RNA.

The first report on the successful transplantation of mammalian nuclei by the example of mice appeared in 1981 (Illmense and Hoppe). In this experiment, the nuclei were extracted from the cells of the internal mass of the blastocysts and transplanted into the zygote of another line of mice with a micropipette. By the same pipette her pronuclei were removed from the zygote. After cultivation of the zygote in vitro to the stage of the blastocyst, embryos were transplanted into recipients - mice of the third line. The resulting 3 offspring, both genotype and phenotype, were identical with the mouse donor line. In 1986, Willadsen transplanted the nuclei on sheep. He extracted unfertilized eggs within 30-33 hours after treatment with their chorionic hormone at the beginning of the hunt. The area of the pellucida above the polar body of the egg was cut with a thin glass needle. One hour after aging the egg in a phosphate medium with cytochalasin, the polar body with its surrounding cytoplasm was aspirated. In this way, approximately 90% of the eggs were split into two parts with intact cell membranes, each containing approximately half the cytoplasm. Cytological analysis showed that approximately 75% of those halves that were removed from the polar body contained chromosomes, i.e. were the "nuclear" halves of the ovum, while the other halves of the same oocytes did not contain nuclear structures, i.e. were enucleated. Single blastomeres from 8- or 16-cell embryos were inserted under the pellucid zone of the enucleated egg. Fusion of the nucleus with the cytoplasm of the cell was carried out with the help of the Sendai virus or electric current. Cells were enclosed in agar and transplanted into ligated sheep oviducts. After 4-5 days, the development of embryos was evaluated. Stages of blastocyst reached from 33% to 48% of embryos. In this experiment, transplantation of 3 of 4 blastocysts was accompanied by the birth of offspring. These lambs were obtained from embryos in which a blastomer from an 8-cell embryo was transplanted into the enucleated half of an unfertilized ovum. In another experiment, it was achieved by transplanting embryos from 16-cell blastomeres. Prather et al. (1987) transplanted blastomeres from two 32-celled cows embryos to enucleated oocytes after maturation in vivo and in vitro by electrosling. Oocytes recovered 36 hours after the start of the hunt and used as recipients of the nuclei of embryonic cells often reached the stage of morulae or blastocysts than eggs matured in vitro or extracted 48 hours after the start of the hunt. 7 pregnancies were achieved after transplantation of 19 embryos to 13 heifers. At 2 heifers live calves were born. The given data give grounds to believe that with the perfection of the efficiency of the technique of transplanting the nuclei of embryonic cells into enucleated oocytes, it is possible to obtain multiple copies from a single embryo.

 


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