Practical significance of chimeric animals (genetic mosaics)
Describe the methods of obtaining identical twins proposed by researchers
The main prerequisite for the manifestation of natural multiplicity in mammals is the same fertilization of at least two mature oocytes from the follicles of different spermatozoa. Cattle are characterized by a small increase in the number of twins - an average of 0.025. Among twins, sometimes identical twins are found. The probability of occurrence of such genetically identical twins is only 0.01%.
Low rates of double frequency and its heritability do not allow to count on high selection efficiency. Therefore, the combination of methods of copying genotypes of highly productive animals on the basis of separation of early embryos by methods of microsurgery and micromanipulation into two or more blastomeres capable of developing in the course of all ontogenesis, ie, capable of showing their totipotency.
The possibility of micromanipulation with individual embryos was proved as far back as 1936 by G Pincus. He injected single blastomeres from a 2-cell embryo of a rabbit into the oviduct of a false-bred rabbit. Embryos normally developed with differentiation of their cells to the stage of blastocysts (stages 2, 4 and 8 blastomeres, morula stage, blastocyst stage). Then offspring was obtained from rabbits and mice from 2-cell embryos (stage 2 blastomeres) with the destruction of one blastomer by piercing it with a needle. These studies served as the basis for further improvement of the technique of micromanipulation with animal embryos, and in particular for the production of identical twins. The latter is of great importance for the intensification of animal husbandry, since it helps to increase the yield of young animals from one donor and allows obtaining genetically identical twins.
For the first time, S.M. Villadsen was informed about the production of identical twins in sheep by dividing the 2-cell blastomer in 1979. He concealed the blastomeres into two separate cells, the disturbed zone of the pellucida (the formation preventing the scattering of blastomeres, as well as contact with other embryos, foreign cells, leukocytes, spermatozoa and facilitating the passage of the embryo through the oviduct) was clogged with agar. The conclusion of separated blastomeres in agar, which is practically insoluble in the female genital tract, allowed them to survive and develop in vivo. The survival of "halves" of embryos at this stage of development after transplantation to recipients is about 50%. In sheep, 2-cell blastomeres can only be obtained within a very short period of time. Later experiments showed that genetically identical twins can also be obtained from 4- and 8-cell blastomeres by dividing them into two groups. Such "halves" were equally viable, like normal embryos of sheep. It was found that embryos obtained from individual blastomeres of 8-cell improper viability. It is believed that a sharp decrease in the number of cells in the embryo is the main factor that reduces their ability to develop into viable blastocysts.
For the production of identical twins, the technology of concluding into agar of blastomere embryos of cattle was successfully applied. So, in 1981 S.M. Willadsen with his colleagues received calves - identical twins. Studies were performed on 5-6 day old embryos at the morula stage, since cows are more suitable to receive embryos non-surgically. Morules were divided into "halves" or "quarters", enclosed in agar and transferred to the ligated oviduct of anestral sheep for 1-2 days. They were then removed and surgically transplanted to recipients on the 6th and 7th day of the sexual cycle. At the same time, the engraftability of the "halves" was quite high (75%), while this index was much lower in quarters (41%).
W.R. Allen and R.L. Pashen (1984) in experiments on horses proved the possibility of obtaining identical twins in a similar method using 2-8-cell blastomeres.
G.Brem et al. (1983) developed a technology for obtaining identical twins using 6-day-old embryos (morules) obtained non-surgically. Embryos were fixed with pipettes. Using a microscore, a pellucid zone was cut, and then a cut was opened with glass needles. A micropart or glass thread was introduced through the hole to cut the morula into two halves. One half of the embryo was left in its own zone of the pellucida, and the other was placed in the transparent zone of the unfertilized oocyte of cattle. According to the authors, the engraftment of the separated embryos was 60%. Of all implanted embryos, 50% of twin pairs were obtained; every third divided embryo gave a pair of twins.
Subsequent experiments to obtain monozygotic twins were aimed at the use of late morules and blastocysts, since at these development stages for embryos of most animals the protection from the zone of the pellucida is unimportant. S.M. Willadsen and R.A. Godke (1984) separated sheep embryos at the stages of the late morula, early, late and hatch blastocyst into two equal halves with a section of the pellucida zone. At the same time, part of the embryo halves remained within the ruptured zone of the pellucida, while others were transplanted without it.
An effective test for assessing the survival of halves of embryos is cultivation for 2-4 hours between separation and transplantation. Culturing the halves of embryos overnight or more than 12 hours was accompanied by a marked decrease in their survival.
Practical significance of chimeric animals (genetic mosaics)
The concept of a chimera means a compound animal. In the modern term, the term chimera is used mainly in the production of composite organisms in which genetically different cell populations occur from more than one zygote or more than one embryo. The acquisition of chimeras or genetic mosaics is currently one of the promising areas of biotechnology. The essence of such a biotechnological method based on the achievements of cellular engineering and micromanipulation in early embryos is the artificial union of embryos of cells of two or more animals that refer not only to one breed, but also to different breeds and even species. Chimera animals carry signs of different genotypes. This is achieved by combining the blastomeres from two or more embryos or introducing cells from one embryo into the blastocyst cavity of another embryo. All the experimental chimeras of mammals, which are still known in science, are created by methods of aggregation of two (or more) genotypically dissimilar embryos or by microinjection of cells of the intracellular mass of the donor blastocyst into the blastocoel of the recipient embryo.
The first method was called aggregation, the second - injection. Fehilly et al. (1984) received complex chimeric embryos of sheep by combining 2-, 4-, and 8-cell blastomeres. Each of these embryos consisted of an equal number of blastomeres of embryos from 2 to 8 parents. The results of a survey of 48 lambs in 2 months. Age showed that 36 goals. are chimeric by blood tests, by external signs or by both indicators. A year later Butter et al. chimeric lambs were obtained by injecting an internal cell mass isolated from donor embryos into blastocysts of recipient embryos. Brem et al. (1985) obtained chimeras in cattle by joining halves of 5-6 day old embryos. 2 calves from 7 had signs of chimerism. One calf in suit was a chimera of brown Schwick and Holstein-Friesian, although he inherited the blood group from the Holstein-Frisian parents. Another calf was an indefinite chimera. Church et al. (1985) obtained chimeric calves by fusion of morules without a zone of the pellucida. They found that transmission of the chimera of each parental type is random, that is, offspring can develop from cells originating from either embryo or from a combination of embryos. The most revealing is the production of chimeras from the fusion of germ cells of different animal species. It is known that neither a sheep nor a goat bore a hybrid offspring until delivery. As a rule, the fruits of experimental hybrid pregnancy in sheep and goats die by the end of the 2nd month. Fehilly et al. (1984) showed that blastomeres of sheep and goats, enclosed in agar and placed on 4-5 days. in the ligated oviduct of the sheep, can form combined blastocysts that are viable and can develop before the birth of normal offspring
The results of the experiments indicate the possibility of carrying out chimeric embryo transplantation between closely related animal species. Interspecific transplantation can provide invaluable assistance in the preservation of endangered species from extinction, as routine transplantation of embryos may be of little benefit, since female recipients may not always be in sufficient quantities. The technique of obtaining chimeras can find application in breeding animals with desirable economic characteristics, as well as resistant to certain diseases.
Chimeric animals do not transmit the genetic mosaic characteristic characteristic of them to the descendants. Like the heterozygous or hybrid animals, the offspring are splitting, as a result of which valuable genetic combinations are violated. Although chimeric animals maintain economically important signs only for one generation, they can be of great practical interest in the breeding of cattle. For example, it is possible to create chimeric animals combining such characteristics as milk and meat productivity, which are antagonistic and incompatible in one organism. Creating injectable chimeras by introducing certain cell lines into the embryo will improve the immune system and increase resistance to a number of diseases.
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