克隆绵羊遇到的问题及其分析  

Sheep Cloning and Analysis of the Problems Involved

作　　者：雪莲[1] 程磊[1] 张立[1] 白春玲[1] 吴侠[1] 董树华[1] 魏著英[1] 苏广华[1] 焦泽华[1] 段彪[1] 康峰[1] 银凤霞[1] 李俊龙 李光鹏[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010021 [2]内蒙古四子王旗赛诺牧业有限公司,内蒙古乌兰察布市011800

出　　处：《内蒙古大学学报（自然科学版）》2011年第2期196-203,共8页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家高技术研究发展计划(编号:2008AA10Z159);内蒙古大自治区重大项目"转基因羊新品种培育";教育部长江学者特聘教授基金

摘　　要：本研究以体外成熟不同时间的绵羊卵母细胞为胞质受体,对去核、融合、不同激活方法、卵裂率、桑/囊胚率和克隆胚受胎率等作了研究与分析.观察妊娠受体母羊的状态、胎儿体内发育情况,分析影响胎儿发育的原因.结果显示,成熟培养18h、22h、24h卵母细胞的盲吸去核率无显著性差异(84.9%,86.6%,84.3%,p>0.05),但显著高于26h组的去核率(71.7%).离子酶素联合cycloheximide(CHX)对重构胚进行激活,卵裂率显著高于A23187联合6-DMAP的激活方法(84.6%vs 63.2%,p<0.05),但二者处理对桑/囊胚率发育无显著差异(25.5%vs18.7%,p>0.05).作为胞质受体,成熟20h的卵母细胞与成熟24h和26h的卵母细胞相比,不利于重构胚的体外发育,桑/囊胚率差异显著(12.3%vs 25.3%,23.4%,p<0.05).分别以美丽奴、杜泊肉羊的体细胞为供体细胞进行克隆生产,90天以后的妊娠率为10%-17%,发育到期率为10%以上.出生羔羊的体重普遍较大.Effects of manipulation technique aspects such as oocyte cytoplast,enucleation,fusion,and parthenogenetic activation on the in vitro and in vivo development of the cloned sheep embryos were studied.After transfer of cloned embryos into recipient ewes,the developmental status of pregnant ewes were systematically observed and recorded during the whole period.The results showed that there was no significant difference in enucleation rates of oocytes matured for 18h,20h,24h（84.9%,86.6%,84.3%,p0.05）.However,They were significantly higher than that of oocytes matured for 26h（71.7%,p0.05）.The activation protocol of ionomycin and cycloheximide（CHX）combination resulted in a significantly higher cleavage rate than the protocol of A23187 and 6-dimethylaminipurine（6-DMAP） combination（84.6% vs 63.2%）.The morulae/blastocyst development,however,has no significant difference between these two activation protocols（25.5% vs 18.7%,p0.05）.When using 24h-and 26h-matured oocytes for the cloning,the morulae/blastocyst development as significantly higher than that of 20h-matured oocytes（25.3%,23.4% vs 12.3%,p0.05）.Donor cells came from Merino and Dorper sheep,after transfer of the cloned embryos to recipient ewes,10%-17% pregnancies were established at Day 90,and more than 10% of them developed to term in both Merino and Dorper sheep.Unfortunately,most of the lambs were overweight,the possible reasons were analyzed.

关 键 词：细胞核移植 胚胎 体外发育 体内发育 胎儿巨大症 

分 类 号：Q132.41[生物学—普通生物学] Q813.7