Method of enhancing engraftment of zygotes, microinjection genetically engineered design in pigs-recipients

 

(57) Abstract:

The invention relates to biology, in particular to biotechnology for producing transgenic animals. Transplantation of zygotes, microinjection genetically engineered design, carried out in two oviduct on 9-12 pieces in each swine-recipients, the reproductive cycle which is synchronized with the sexual cycle of pigs donor embryos. The method allows more than 20% increase gestation in pigs-recipients and almost 2 times the accepted embryos. table 1.

The invention relates to the field of biology, in particular to biotechnology for producing transgenic animals, and can be applied to improve the efficiency of producing transgenic pigs.

Great attention to the pig as the object for transgenes, due to the fact that the pig is a multiple animals with relatively short reproductive cycle. From it you can get a large number of fertilized oocytes that are required for introduction of foreign genes in them, and to get from one recipient of multiple children, developed from zygotes, microinjection these genes, which is an important condition for successful work on transgenes.odstv, improved genotype pigs and acceleration of the breeding process in the direction of increasing productivity, resistance to disease and improve product quality by using transgenesis has great economic importance. In addition, calculations show that because of its mnogoplodnoy and Skoropadsky pig can be useful for producing transgenic animals superproducers human biologically active substances of pharmacological purposes.

In recent years, intensive research aimed at clarifying the possibility of transplantation of organs and tissues from animals to humans (xenotransplantation). Taking into account the anatomical and physiological similarity and proportionality in some organs of the pig (heart, kidneys, and other) with human organs, and the absence of common infectious diseases, researchers tend to assume that when a specific genetic interference in immune processes in organs of transgenic pigs can be used for xenotransplantation their man. Thus, thanks to mnogoplodnoy and Skoropadsky pig becomes one of the main objects technology transgenes.

Up to the present in the stock of structures in the pronuclei of zygotes, including various micro-manipulation (centrifugation, puncture cytoplasmic and nuclear membranes, the introduction into the pronucleus of foreign DNA in a buffer solution, and so on), which has some negative influence on the development of microinjection zygotes and, accordingly, their settling down in transplantation pigs-recipients.

Transplantation of zygotes, microinjection genetically engineered design, swine-recipients is one of the main stages of the technology for producing transgenic pigs, which largely depends on the settling down of the embryos, the calving and therefore the overall effectiveness of the technology.

There are two methods of transplantation of embryos of pigs.

The first method embryo transfer pigs-recipients is that the embryos in the late stages of development (morula and blastocyst) in the amount of 15-18 pieces are transplanted into one uterine horn surgical method using a special catheter.

Transplantation of embryos of pigs in one uterine horn is based on the prescribed 60-ies of the fact, proving that the embryos of pigs have the ability to migrate from od. the property porcine embryos later began to use in the transplantation of embryos preimplantation stages of development of animal-recipients. Conventional pigs-recipients transplanted embryos in one uterine horn, believing that the embryos themselves evenly distributed in the horns of the uterus due to the effect of migration. Embryo transfer in one horn of the uterus is less time-consuming and requires less time. Gestation pigs-recipients after transplantation of embryos in the late stages of development in one uterine horn is 50-60%, and the settling down of embryos 20-25% (Hunter, R. H. F., Polge C. and Rowson L. E. A. - The recovery, transfer and survival of blastocysts in pigs. - J. Reprod. Fert. 1966, v. 14: 501-502).

The second method. The closest to the technological nature of the present invention is a method of transplantation of zygotes, microinjection genetically engineered design (prototype). In this way the pigs donor cause superovulation and produce the extract from the fallopian tubes of embryos that are on the stage of the zygote, then the pronuclei of zygotes microinjected genetically engineered design and then microinjection zygote transplanted in one of the fallopian tubes pigs recipients, the reproductive cycle which is synchronized with the sexual crantzii embryos animal-recipients transplanted't blastocyst as the zygote, which have been genetically engineered design, then they are not in the uterus and oviduct, and in quantities far exceeding the number of blastocysts grafted onto the horn of the uterus in normal embryo transfer (30-40 vs 15-18, respectively), because whereas the decreased viability of some zygotes, which occurs in the process of manipulation associated with microinjection into them genetically engineered design. As with transplantation of normal blastocyst, zygote, microinjection genetically engineered design, is introduced into one oviduct pigs recipient into believing that the developing embryos will be able to migrate to the second horn of the uterus and evenly distributed in the horns of the uterus. However gestation pigs recipient and accepted embryos in this way, transplantation remains very low (30-40% and 5-7%, respectively) (K. Spinsmann, G. Brem - - Embryotransfer beim Schwein im Rahmen von Gentransferprosrammen - Tierarztl. - Prax. Suppl. 4, 21-25, 1989).

In this study, one group (I gr.) pigs-recipients transplanted less than 40 microinjection embryos, and the second group is more than 40. Pregnant steel 25% of the recipients of the first group and 46% in group II. However, the settling down of the embryos was higher in pregnant recipien the t number of zygotes, transplanted pigs-recipients, showed that increasing the number of transplanted embryos from 10 to 40 increased the number of pregnant recipients from 20% to 43%. But the accepted embryos have decreased from 33.3% for the transplant 10 embryos to 13.3% for the transplant 31-40 embryos. A similar decrease in engraftment of embryos was observed in terms of all recipients from 8.9 to 5.7% for the transplant 11-20 and 31-40 microinjection zygotes, respectively. A lower percentage of engraftment of embryos in the transplantation of a large number microinjection zygotes (over 30) apparently due to increased mortality of embryos at the stage of elongation, when a small area of the horns of the uterus accumulates a large number bongiovanni embryos length up to 1 meter. And therefore the highest percentage of engraftment of embryos observed in the transplantation is not a very large number microinjection zygotes. In this case, after the death of a specific part of the embryo in the early stages of development, caused by a variety of manipulations associated with microinjection into them genetically engineered structures, stages of elongation reaches fewer embryos, which saved the best condition is usemost zygotes, microinjection genetically engineered design, in pigs-recipients to improve the overall efficiency of producing transgenic pigs.

This goal is achieved by the fact that in the proposed method transplantation zygotes, microinjection genetically engineered design, carried out in two oviduct on 9-12 pieces in each swine-recipients, the reproductive cycle which is synchronized with the sexual cycle of pigs donor embryos. In the known method transplantation microinjection zygotes produced in one oviduct in the amount of 30-40 pieces.

Example. To obtain a large number of zygotes in pigs donor caused superovulation. The group of pigs that were 12 to 15-th day of the sexual cycle (counting the first day of the previous hunting zero day), in the same day twice with an interval of 8-10 hours) was subcutaneously injected one of the drugs of prostaglandin F2the dose recommended for pigs according to the instructions. In 24-36 hours after injection of prostaglandin pigs intramuscularly once entered one of the drugs serum foals mares (FLC) in the dose of 1200-1500 I. E. on the head, and through 72-80 hours after administration of gonadotropin FFA animals once intramuscularly injected Horonite and after 16-18 hours were spent removing the embryos at the stage of the zygote. To synchronize reproductive cycles pigs-recipients were treated with hormones in the same way that pigs donor, except that the dose of FFA was reduced to 600-700 I. E.

Embryos at early stages of development were removed by washing of the fallopian tubes in animals donor environment Dulbecco enriched with 4% fetal bovine serum.

Morphological evaluation of embryos of pigs was performed under an inverted microscope equipped with optics of Namestovo in DMEM with HEPES, enriched with 20% fetal bovine serum. To facilitate visualization of pronuclei in zygotes and cores in the 2-4-cell embryos would centrifugation at 10.000-13.000 g for 5-7 minutes, providing the offset lipid and pigment inclusions to one of the poles of the embryo.

Microinjection of genetic engineering in the pronuclei of zygotes were made of glass injection needle with an outer tip diameter of 1.5-2 μm at the facility, including inverted microscope type ICM-405 with optics differential interference contrast (DIC) and a set of manipulators and injectors. The accuracy of the introduction of exogenous DNA solution was determined by the increase in rassledovanie human gene granulocyte-colony stimulating factor under the promoter of the gene S1-casein cattle. The volume of foreign DNA introduced into the pronuclei was 1-2 dps with a concentration of 4-6 ng/ml, which corresponded to 400-600 copies of genetically engineered design for one injection.

Transplantation microinjection embryos pigs-recipients conducted in ampullary part of the oviduct from the fringe using a special plastic catheter. Pigs-recipients in the control group of the zygote in the amount of 20-25 pieces were transplanted into one oviduct, and the animals of the experimental group in both oviduct 10-12 zygotes in each oviduct.

The analysis of the experimental results (see table) shows that the percentage of gestation was significantly higher in pigs-recipients who transplanted microinjection zygote in both oviduct, i.e. bilateral transplantation (66,6% compared to 45.4%), while almost the same number of zygotes, transplanted one recipient (22,32,2 and 21,32,4). A similar pattern was observed for the engraftment of embryos, both in terms of all recipients (10.1% versus 3.4 percent), and single pregnant recipient (15,2% against 7.5 per cent, respectively).

This increase in the percentage of pregnancy and engraftment of embryos in pigs recipient is achieved sideline embryos in the uterus horns, what creates the most favorable conditions for development than unilateral transplantation.

Thus, the proposed method transplantation zygotes, microinjection genetically engineered design, allows more than 20% increase gestation in pigs-recipients, 2 times - accepted embryos, and the output of offspring, and hence the overall effectiveness of the technology for producing transgenic pigs.

Method of enhancing engraftment of zygotes, microinjection genetically engineered design, in pigs-recipients, including synchronization sexual cycles pigs donors and recipients and the transplantation of these zygotes in one of the fallopian tubes pigs-recipients, characterized in that the transplantation of zygotes pigs-recipients spend in both the oviduct, and in each oviduct transplanted on 9-12 zygotes, microinjection genetically engineered design.

 

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FIELD: animal science.

SUBSTANCE: the present innovation deals with dynamic loading onto cardio-vascular system in animals. Selection should be carried out by the following parameters: , ΔT3 and Δn, where ΔT1 - the time for pulse increase at running, ΔT2 - the time for pulse stabilization after running, ΔT3 - the time for pulse increase after running, Δn - the increase of pulse frequency after running. One should select animals into milking herd at the following values; ΔT3 ≤ 10 sec, Δn ≤ 10 beats/min. The method enables to present perspective evaluation of lactation capacity in animals.

EFFECT: higher efficiency of selection.

1 dwg, 1 ex, 1 tbl

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