牛卵母细胞孤雌激活及马-牛异质克隆胚构建

Parthenogenetic Activation of Bovine Oocytes and Heterogeneous Embryo Reconstruction by Horse Skin Fibroblasts and Bovine Oocytes

首先用不同的激活剂孤雌激活体外成熟培养的牛卵母细胞,经试验获得:离子霉素、A23187和7%乙醇联合6-DMAP可有效地激活牛卵母细胞,并支持其发育到囊胚,但离子霉素激活效率显著优于其他两种(P<0.05);以10%FBS+SOFaa+颗粒细胞为发育体系培养激活的成熟牛卵母细胞可得到较高的卵裂率和囊胚率(72.30%,14.91%)。其次,通过体外培养成年马皮肤成纤维细胞,将获得的成纤维细胞经血清饥饿培养后,作为核供体移入去核牛卵母细胞透明带下,电融合后,能得到融合的马牛重构胚,在交流电脉冲起始电压20V,持续时间10s,频率0.2MHz,结束电压15V,2次脉冲和融合间隔为0.125s的条件下,当融合电压为2.0kV/cm,脉冲时程为40μs时,重组胚的融合率和卵裂率最高(52.27%,71.74%)。

Firstly, in vitro matured bovine oocytes were activated by three different activators. The results showed that matured bovine oocytes were efficiently activated by Inomycin, A23187 and 7% ethanol alone followed by 6-DMAP, respectively, and developed to blastocyst phase, but the cleavage rate and the blastocyst rate of matured bovine oocytes activated by inomycin-6-DMAP were significantly higher than those of matured bovine oocytes activated by the two others （P〈0.05）; When the activated oocytes were cultured in SOFaa added with 10% FBS and cumuluses, a higher cleavage rate and blastocyst rate were achieved （72.30%, 14.91%）. Secondly, horse skin fibroblasts were cultured in vitro and induced to quiescence stage （G0 phase） by serum starvation culture. The cells were injected into enucleated bovine oocytes and the reconstructed heterogeneous embryos could be obtained. The cell fusion was induced by 2.0 kV/cm for 40 μs under the condition of 20 V start-voltage, 10 s duration, 0.2 MHz frequency, 15 V end-voltage and an interval of 0.125 s, fusion rate and cleavage rate of reconstructed embryos reached the peak values （52.27%, 71.74%）.