| Transgenic technology is the use of molecular biology techniques, based on genetics and recombinant DNA technology will be isolated from the exogenous gene (restructuring) import animal zygote or early embryonic cells, to integrate into the host cell genome, to pass on to the next generation of a biological technology. Its purpose is to transform of biological genetic material, get some in good character quality, nutrition and consumption characteristics of animals. Transgenic animals has become one of the fastest growing and most popular areas now. Transgenic animal technology have a wide range of applications in the establishment of animal models to study gene function and treatment of many human diseases, animal breeding for disease resistance, xenotransplantation, biological pharmacy. In animal husbandry, transgenic technology opened up new avenues for the transformation of animal species, which have new promote growth genes that does not exist, and through transgenic cloning it will help to cultivate new varieties of animals. Although transgenic animal technology has important application potential in a wide range of areas, but the low rate of success transgenic animals, the integration of foreign genes, the effect of instability, the safety of genetically modified animals, etc that so severely restricte the further development and use of this technique. Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious infectious disease that can cause respiratory disease of adult reproductive failure of sows and piglets and brought huge economic losses in the production of China’s pig industry. In this study, by the use of RNA interference (RNAi) gene silencing technology and somatic cell nuclear transfer technology, we product anti-PRRSV shRNA transgenic cloned embryos in vitro, and focuse on the optimization and improvement of get cell transgenic cloning technology application, analyses the technology method there may be some problems; in order to improve the efficiency of the pig cloned embryo production in vitro. The experiment is mainly to get mature oocytes as the outside material, research get cells cloned embryo in vitro production of key technologies, set up more perfect technical system, based on the stable production of pigs against PRRSV transgenic cloned embryos. The results are as follows:Experiment1. Effects of hormone on the maturation of porcine The porcine ovarian acquisitied from slaughterhouse. Cumulus-oocytes composite collected from2-5mm follicles COC by aspiration, basic culture medium used for TCM-199and add5%porcine follicular fluid (PFF),10%fetal bovine serum (FBS),10ng/mL. epidermal growth factor (EGF) and0.1mg/mL L-cysteine. The COCs medium of control group, extra10IU/mL at horse serum gonadotrophin (PMSG) and10IU/mL human chorionic gonadotropin (hCG) cutured for42h. The COCs of experimental group cultured with the control group, after20h moving hormone-free TCM-199based fluid and cutured22h. Statistics oocyte maturation rate of the control group oocyte maturation with the first polar body discharge rate was73.23%, while the test group pbI discharge rate of86.48%, the difference was significant (P<0.05). Experiments show that the first add10IU/mL PMSG and10IU/mL hCG hormone TCM-199basal medium and cultured for20h, and then transferred to hormone-free TCM-199basal medium cultured22h mature training program obtain better oocyte maturation rate.Experiment2. Effect of cell cycle synchronization on the development ability of pigs transgenic cloned embryosTransgenic pig fetal fibroblast cells as donor cells, the test group used serum starvation and contact inhibition to induce the transgenic donor cells into the G0/G1phase, maturation oocytes of pig as recipients for somatic cell nuclear transfer test. Results show that treatment for the same period of the cell cycle has some influence on the development of transgenic cloned embryos. Blastocyst development rates of the experimental group were26.79%and21.78%, respectively, which were higher blastocyst rate (16.67%), but among the three groups the difference was not significant (P>0.05).Experiment3. Effect of preparation methods on the development of pigs transgenic cloned embryosThe experiment was divided into three groups (fresh digestion refrigerated at4℃, freezer-70℃), compare the effect of preparation methods on the development of pigs transgenic cloned embryos. Results show that the blastocyst development rate of4℃refrigerated group and fresh digestion group were higher than the-70℃frozen group (20.17%vs7.792%;20.56%vs7.792%, P<0.05). However there were no significant differences between4℃refrigerated group and fresh digestion group (P>0.05).Experiment4. Effect of different activation treatment on the development of pigs transgenic cloned embryos The experiment by pig fetus fibroblast as nuclear donor, builded reconstructed embryos. Under the ideal parameters1.5kV/cm,40p.s and1DC, We had studied the effection of electrical activation and the activation of6-DMAP chemical activation on the development of porcine embryos. Results show that both pure use electric activation or electric activation add6-DMAP process, cleavage rate and blastocyst development rate between the two groups had no significant difference (69.39%to70.53%,25.51%to69.39%, P>0.05).Experiment5. Effects of different transgenes donor cells on the development of pigs transgenic cloned embryosThe experiment by pigs fetal fibroblasts (pFF) and ear fibroblast cells (pEF) as the nuclear transfer donor, respectively, for somatic cell nuclear transfer, compared their influence on transgenic cloned embryos, and Results show that although the cleavage rate of two groups had no significant difference (77.31%to80.00%, P>0.05), but the pFF of blastocyst development rate (23.71%) was significantly higher than that of pEF group (15.88%).Experiment6. Effect of TSA on the development of pigs transgenic cloned embryos0nM and50nM TSA handling nuclear donor cells, the blastocyst rate (20.47%and17.67%) was significantly higher than100nM TSA group (10.38%), and between0nM TSA group with50nM TSA group blastocyst, the development was no significant difference (P>0.05).With50nM TSA treatment transgenic cloned embryos for24h, the blastocyst development rate (24.82%)can be obtained; As if the TSA treatment for nuclear cells and embryos, the blastocyst development rate (28.09%) was significantly higher than they would without the TSA treatment control group (P<0.05). |
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