| Fibroblasts were separated from the boer 21#(about 1g) ear tip and cultured in vitro for donor cells to P11P12, and M II stage oocytes were got from delta of the Changjiang River through superovulation.When the cells confluence about 80%, a part was selected for synchronization to M stages: the frist step, treat with Thymidine for 16 hours , then recover culture for 5 hours , the third step was treat it with Nocodazole for 7-8 hours, the last was 30 minutes before transfer select the cells with digestion ; the other was treated with nothing and select for nuclear transfer. The reconstructed embryos which were produced by electro-fusing the donor cells into the recipient cytoplasts were cultured for 4-6 hours before activated. After activated , embedded these embryos with 1% agarose for two times , then transferd them into the ligated oviduct of synchronized recipient goat. Results: (l)The difference between the fusion rate and the development rate of reconstructed embryos which were treated with Thymidine and Nocodazole(40. 57% 41. 02%) and which were treated with nothing(44. 64% 42.65%) were not obvious; (2)The reconstructed embryos of boer goat somatic cells and the M II stage enuclear oocytes were develop to term; (3)The donor cells which were used for transfer were cultured in vitro to P11桺12, these suggest after long time culture in vitro boer goart cells also have totipotent potential of development; (4)In theory, The donor cells which were treated with chemistry regent at M stage used to nuclear transfer can get cloned goat; (5)From the phenotype , suggest that term is a cloned boer goat; (6)The alalyse of microsatellite DNA further proved that term is a cloned boer goat. |
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