Pig Somatic Cell Nuclear Transfer Transgenic Technology Research

Pig Somatic Cell Nuclear Transfer Transgenic Technology have great dominance and potential as models for human disease and as human xenotransplantation, and it also may be used in pig breeding, even the production of "green pig".therefore this research has been a research focus. In this study, the establishment of efficient pig somatic cell nuclear transfer transgenic technology system for the purpose of, and tries to build construction of embryo transfer phytase gene. For the future production of "green pig" lay a solid foundation.The objective of the experiment was to compare the correlation of three ways to determine matured MⅡoocytes, which are observing the first polar body, oosight imaging system observation and hocchst33342 staining, and to evaluate the effects of pOCCs, pOECs and pCCs monolayer co-culture system on porcine cumulus cells denuded oocytes during in vitro maturation and parthenogenesis respectively. The results show that:(1) Determining the outcome of oocyte maturation, the result of oosight imaging system is significant correlation with that of the hocchst33342 staining(R=0.973, P<0.01, N=90); (2) Comparing with pOECs group, the rate of Dos maturation in pOCCs group in vitro maturation is significantly higher (52.5±0.30% vs 43.8±2.18%, P<0.05), and the rates in the pOCCs group and pOECs group are both significantly higher than that in the other groups(P<0.05); After Dos culturing in vitro maturation in three kinds of cell monolayer co-culture, GSH content significantly increased(P<0.05), pOCCs group had the highest content(7.1±0.32 pmol/egg); (3)Mature Dos after parthenogenetic activation, the Cleavage rate and Blastocyst rate in pOCCs group and pOECs group are significantly higher than in the other two groups(P<0.05).Established Fengjing pig 35d fetus fetal fibroblast cell line, adult ear fibroblast cell line, oviduct epithelial cells, and isolated and cultured porcine granulosa cells in follicular fluid. And compared the method of liposomeDNA content, liposome concentration and incubation time on transfection efficiency. The results showed that, The four cell lines by 24-well plate cell confluence 85%-90%, 1.0μg DNA and the amount of liposome 2.4μL, 4h incubation have most efficient with YM6552 plasmid transfection. The four cell lines require the same liposome-mediated conditions. Which fetal fibroblasts transfected with the highest efficiency.Between somatic cell nuclear transplantation transgenic, We are mediated by LipofectamineTM 2000 transfection of four for nuclear cells line with YM6552 plasmids. And using G418 screening them. By screening out stable transfection for nuclear cells line with YM6552.Got the expression of green fluorescent protein in reconstructed embryos. When building Reconstruction embryos, compared for four types of transfected cells Impact on the in vitro development of reconstructed embryos. The results show that, fetal fibroblast cells were able to get the highest cleavage rate and blastocyst rate after Been transfected YM6552 plasmid.