Cloned Sheeps From Adult Skin Fibroblast Cells

This study attempted to produce cloned sheep using sheep skin fibroblast cells as donor karyoplasts and enucleated sheep oocytes as recipienf cytoplasts. established the ovine skin fibroblast cell line, compared the effect of different methods of parthenogenetic activation on in vitro matured ovine oocytes, and further studied the effect of the condition of donor cell and factors of cell fusion on ovine somatic cell nuclear transfer.Ovine skin fibroblasts were dissociated and cultured in vitro. The primary cells were obtained by explant tissue culture and enzyme digestion, and the former method was more easier to obtain ovine fibroblasts than the letter. The growth curves of the 5th-passaged and 10th-passaged cell were drawn, and there was no significant difference in growth velocity between these two passaged cell. The karyotypes of the 5th-passaged and 10th-passaged cell were analyzed, and about 90% of the 5th-passaged cells and 84% of the 10th-passaged cells maintained diploid karyotype (2n=54).The in vitro matured ovine oocytes were panthenogenetically activated by electroactivation, 5mM Inomycin for 5 minutes, 7% ethanol for 7minutes combined with 2mM 6-DMAP for 4 hours respectively.The cleavge rates were 60.93%,79.96%and78.28% respectively, the percentage of blastocyst were 5.27%, 18.51%and 15.80% respectively.Ovine embryos were reconstructed from not-starved, serum-starved, and cryo-preservated donor cells. The percentage of fusion were 45.79%,43.78%and43.76% respectively;the cleavge rates were55.7%,52.98%and43.48 % respectively,the percentage of morula were 44.42%,42.48%and41.16% respectively.A tolal of 412 reconstructed embryos were transferred into 54 surrogate sheeps, 11 were pregnant.