The Production Of Cloned Sheep From Adult Skin Fibroblast Cells

In this research , different parthenogenetic activationon methods of in vitro matured ovine oocytes were compared, which laid a foundation for activation of followed reconstructed somatic cell nuclear transfer embryos of sheep; The effects of different fusion voltage on development of reconstructed embryos was studied; And the lamping rate of ET for various developmental stages were compared.Under the condition of 37℃,saturated humidity and 5%CO2,The sheep skin fibroblasts were cultured in DMEM/F12 with supplemented of 10% FBS using direct tissue culture. It can establish the sheep skin fibroblast line effectively and be confirmed that the sheep skin fibroblasts basically corresponds growth characteristics of cultured cells by growth curve assay and chromosome counts.Matured sheep oocytes were efficiently activated by electroactivation, 5mM Inomycin for 5 minutes, 7%ethanol for 7minutes combined with 2mM 6-DMAP for 4 hours, The cleavage rates were 57.68%,78.90%and75.47%respectively,the percentage of blastocyst were 18.31%,41.95%,37.11%respectively. Inomycin for 5 minutes combined with 2mM 6-DMAP for 4 hours is the best.Different development stage reconstructed embryos(just fusion,2～16 cells,morula\blastocysts)were used in the embryo transfer , The percentage of pregnancy were 14.4%/,0,3.4% The cloned embryo of just fusion was significant higher than the other two.