Factors Affecting Reconstructed Embryos Formation In Porcine Somatic Cell Nuclear Transfer

The production of transgenic cloned pigs with site specific alterations would have great benefits in agriculture as well as human therapeutic applications. Although dozens of nontransgenic and transgenic pigs have been born after somatic cell nuclear transfer, and several new methods have been introduced, the overall efficiency remains low due to the poor in vitro embryo development. This research aim to establish an efficient system for producing pigs by somatic cell nuclear transfer(SCNT).Effect of hormone,pFF and EGF on maturation of porcine oocytes have been investigated. With regard to the development of porcine nuclear transfer embryos, systematical study had been carried on factors including donor cells,co-cultural systems with Vero cells and energy substrate. At the same time, this study pertain to investigating the effect of TSA(a histone-deacetylase inhibitor) on EGFP expression in transfected cells and embryonic development after SCNT, providing theoretical significance for producing transgenic pigs.Hormone,PFF and EGF have positive effects on maturation of porcine oocytes. When treated with 10 IU/ml PMSG,10 IU/ml hCG for first 24h,oocytes have higher rate of maturation than other treatments(P<0.05). When pFF was added into maturation medium, rate of maturation in 10%pFF treatment was significantly higher than other treatments(P<0.05). When oocytes were treated with EGF, the rate of maturation was significantly higher in 10ng/ml group than that in 0 ng/ml and 5ng/ml groups(P< 0.05); there was no significant difference between lOng/ml group and 15ng/ml group(P>0.05).Yorkshire fetal fibroblasts can be used as nucleus donator in somatic cell nuclear transfer due to the characteristic of easy to be separated and rapid growth velocity, strong passage capability, more amplification generations in vitro culture. Chromosome analysis was performed in different cell passages. Normal rate of chromosome nuclear type can reach to 80% when cultured up to passage 7, which satisfied the requirement of somatic cell nuclear transfer. Smooth cells or rough surface cells were used as donor in SCNT. Smooth surface cells led to a higher fusion rate. With the number of passage of donor cells increasing, the rate of blastocysts formation decrease.The Vero cells line was derived from the kidney of a normal, adult, African green monkey on March 27,1962, by Y. Yasumura and Y. Kawakita at the Chiba University in Japan. Despite the abundance of literature on the positive effects of Vero cells on some mammalian embryonic development, there are few reports about co-culture system with Vero cells throughout the culture interval of porcine embryos.This experiment was conducted to study the effect of Vero cells in embryo co-culture system on the efficiency of blastocyst production in pig. The result showed that groups co-cultrued with Vero cells achieved significantly higher blastocyst formation rate compared with control(P<0.05).Co-culture with Vero cells improve the development of porcine embryos in vitro. This is the first time that Vero cells were used as feeders in producing cloned pigs.This study also investigate the effects of pyruvate and lactic acid on the earlier development of porcine embryos.5.56 mmol/L glucose in culture medium (NCSU-23) was replaced with 0.2 mmol/L pyruvate and 5.7 mmol/L lactic acid, namely mNCSU-23. Parthenogenetic embryos and nuclear transfer embryos were transferred into NCSU-23 or mNCSU-23 medium according to the experimental design. Parthenogenetic embryos and nuclear transfer embryos were evaluated for the numbers of 5-8 cells stage on Day 2. Blastocyst rates and the numbers of nuclei in the blastocyst were determined on Day 6. We observed that the rates of blastocysts formation in mNCSU/NCSU treatments on Day 6 were significantly higher than control, with mNCSU-23/mNCSU-23 treatment having the lowest rates of blastocysts formation on Day 6 (P<0.05). Our results have demonstrated that replacing glucose with pyruvate and lactic acid during the first part of IVC may be beneficial to the development of the porcine embryo.At present, there are many methods for exogenous gene to introduce eukaryotic cell, such as Viral infection, Pronuclears microinjection, DEAE-glucosan infection protocol, Electroporation, Lipofectamin-mediated DNA transfection method and so on.Liposome infection protocol is simple, doesn't needs expensive instrument, can transfection a great deal of cells and less harm to cell. So in this study, pEGFP-Cl vector was introduced into fetal fibroblast of Yorkshire by lipofectamin-mediated DNA transfection methods, transfection parameters were optimized. Lipofectamin 4μl, plasmid vector DNA 2μg, stable transfection 6 hours are optimized condition for transfection. Yorkshire fetal fibroblasts were transfected with pEGFP-Cl plasmid. After selecting with G418, stable transfected cell line was gotten. With the increase of passage number, EGFP expressed cells significantly decreased. Then transfected cells, donor cells for SCNT, were pre-treated with TSA, with the untreated cells used as control.Expression of EGFP in donor cells and re-constructed embryos was detected when exposed to blue light. Results have shown that percentage of EGFP-positive cells significantly increased when the transfected cells were treated with 50nM TSA and percentage of EGFP embryos on day 2 from 50 nM TSA treatment was significantly higher than control groups and increaed transgene expression continues to at least the morula stage. In addition, it appeared that the cytotoxic effect of TSA on the transfected cells was dose-dependent. Most of the cells died when TSA consentation was over 50nM(P<0.05). The cells changed morphologically at 24 h after TSA treatment. TSA-treated cells elongated in size and were vague in outlines.