【摘要】 以 - 35℃低温冷冻保存 3个月的Vari Fe 8mos和Fahru Fe 9mos死亡牛胎儿皮肤上皮细胞 ,为核供体细胞 ,应用点击去核方法 ,和 - 30 0mmHg、2 %CO2 、8%～ 10 %O2 、38.5℃和 10 0 %湿度负压气相培养系统 (负压组 ) ,进行了克隆牛试验研究 ,并与吸引和挤压及常压培养法进行了比较。结果表明 ,采用点击去核法对体外成熟培养18h、2 2h、2 4h的卵母细胞进行去核 ,去核成功率分别为 90 .0 %、75 .8%和 5 5 .6 %,18h组显著高于 2 2h组 (P <0 .0 5 ) ,极显著高于 2 4h组 (P <0 .0 1) ;而且 ,点击去核法去核率为 90 .0 %,极显著地高于吸引法的 6 8.3%(P <0 .0 1) ,与挤压法去核率 88.3%差异不显著 ;采用点击去核法囊胚发育率达 34 .1%,显著高于吸引法的 18.0 %(P <0 .0 5 ) ,与挤压法的 2 0 .9%差异不显著。负压组重构胚的卵裂率达 70 .0 %,显著高于常压组的 6 3.8%(P <0 .0 5 ) ,负压组囊胚发育率达 35 .8%,极显著高于常压组的 2 3.2 %(P <0 .0 1) ,负压组克隆囊胚细胞数为 10 8± 3.3个 ,极显著多于常压组的 98± 3.3(P <0 .0 1)。将第 6天的克隆桑椹胚和囊胚每 2～ 3枚装入 1支 0 .2 5ml塑料细管中 ,移植给同期发情处理的 5头受体奶牛的黄体侧子宫角中 ,最终获得 2头牛妊娠 ,并于 2 0 0 1年 11月 3日和 11月 6更多还原

【Abstract】 In this study, oocytes (MII stage) were enucleated by point-hit method, loaded with epidermal cells (as donor cells) derived from frozen bovine fetus(Vari-Fe-8mos and Fahru-Fe-9mos) which had been stored at -35℃ for 3 months. Results of the group of the reconstructed embryos being cultured under 38.5℃, 2%CO 2, 8%-10% O 2, 100% humidity and -300 mm Hg negative air pressure(negative air pressure group) were compared with that under normal air pressure(normal air pressure group), the different enucleating methods were also compared. The results showed that the enucleating rates of oocytes (enucleated by point-hit method) being matured in vitro for 18h, 22h, 24h were 90.0%,75.8% and 55.6% respectively, the enucleating rate of oocytes being matured for 18h was significantly higher than that for 22h (P<0.05) and that for 24h (P<0.01); Further, the enucleating rate of oocytes being enucleated by point-hit method was 90.0%, significantly higher than that by sucking method (68.3%,P<0.01), not different with that by squeezing method (88.3%).The blastocyst rate of oocytes enucleated by point-hit method was 34.1%, significantly higher than that by sucking method (18.0%,P<0.05) and not significantly different from that by squeezing method ( 20.9%). The cleavage rate of reconstructed embryos being cultured under negative air pressure was 70.0%, significantly higher than that under normal air pressure 63.8% (P<0.05), the blastocyst rate of those under negative air pressure was 35.8% significantly higher than that under normal air pressure (23.2%,P<0.01). The cell number of blastocyst in negative air pressure group was 108±3.3, significantly more than that in normal air pressure group (98±3.3,P<0.01). Every a 0.25 ml plastic straw was loaded with 2-3 fresh cloned embryos (day 6) that developed into morulaes or blastocysts. The cloned embryosefter synchronization of estrus. At last, 2 recipient cows became pregnant. On November 3 and 6, 2001, 2 female beef calves (named as "Kang Kang" and "Shuang Shuang") were delivered respectively. The results of analysis by microsatelite DNA genotyping show that those two calves were corresponding to Vari-Fe-8mos& "Kang Kang", Fahru-Fe-9mos&"Shuang Shuang", respectively. This experiment shows that it has certain advantage and practicability for cloning cattle by using epidermal cells ( derived from frozen bovine fetus which had been stored at -35℃ for 3 months) as donor cells, enucleating by point-hit method, culturing under the system of negative air pressure.更多还原