Preparation Of Embryo Of Transgenic Somatic Cell Nuclear Transfer

Ovaries used in this thesis are collected from the slaughter hall of Pengcheng food company Ltd in Shunyi district of Beijing. The ovaries were carried to laboratory within two to three hours in the temperature environment of 30-35℃. The oocytes were cultured for 42-44h and then collected for parthenogenetic activation. The experiment was conduced to grope the electrical field strength and pulse width of porcine oocyte parthenogenetic and the effect of stage-culture method of osmotic pressure on parthenogentic embryo development. Afer 42-44 h of in vitro maturation, porcine oocytes were activated under the condition of 2.1 kv/mm,2.3 kv/mm,2.5 kv/mm and 30 ms,60 ms,90 ms. Using the parameter of 2.1 kv/mm and 30 ms for parthenogenetic activation and then the oocytes were cultured in PZM-3 with the osmotic pressure of 271 mOsm,280 mOsm,290 mOsm,302 mOsm each other. Afer electrical activation, the oocyte were cultured in PZM-3 with 2 mmol/L 6-DMAP for 4-6 h and then cultured in PZM-3 without 6-DMAP for the last time. The results showed that 1)there were no interaction between electrical field strength and pulse width(P>0.05). Both of the three electrical field strength have no significant effect on cleavage rate(P>0.05). The effect of pulse strengh of 30 ms,60 ms,90 ms on cleavage rate took on the tendency of descending. However, the blastocyte rate of each test group had no significant difference(P>0.05); 2)Cultured for 48 h under the condition of osmotic pressure of 290-310 mOsm in PZM-3,the cleavage rate of parthenogentic embryos were significant higher than other groups(P<0.05), however, there were no significant difference of blastocyte rate(P>0.05); 3)6-DMAP has no significant effect on the cleavage rate of parthenogentic embryo(P>0.05) but the blastocyte rate was significantly increased(P<0.05). These results indicated that porcine oocyte activation needs high electrical field strength(2.1-2.3 kv/mm) but the pulse width must be short(30 ms) ; Hypertonic culture for 48 h and second activation with 6-DMAP will promote the later stage development of parthenogentic embryo.Red fluorescence protein(RFP) gene was used to mark WZSP foetus fibroblast for detection signal of technology platform of porcine somatic clone. We can distinguish the transgentic pig form none just utilizing fluorescence excitation dispensing with molecular biology detection. Otherwise, in the process of embryo production it ensures the real time observation. So that we can evaluate the feasibility of technology and method. It is very important for production of transgentic pig. In this article we have used foetus fibroblast from inbred strais of WZSP. The Lipofectamine? 2000 lipidosome transfection kit was introduced to transfer RFP to porcine foetus fibroblast. The positive rate of cell transfection is 27.52%(79/287) achieving the demand of this research. Both of positive cell clone screening methods are sufficient for next research. Our study has verified that RFP transgenic WZSP foetus fibroblast obtaind in this research is usable and the technology process and method is scientific and reliable. During the time of RFP cell screening we used normal none transgenic WZSP foetus fibroblast and EGFP transgenic foetus fibroblast(Zhao jianguo offering,Chinese Academy of Science) to clone. Three index of fusion rate, cleavage rate and blastocyte rate were used for adjusting and optimizing each section of technology platform. The average acquisition rate of each ovary has been elevated and the period of micromanipulation has been decreased. And then the efficiency of somatic cell clone has been improved(cleavage rate ,50% ; blastocyte rate ,12%-15%). We proceeded with the transplant experiment simultaneously. Up to now we have transplant four sows and follow-up experiment is in progress. The detection of transplant efficiency is pending.