山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.

STUDY ON THE CLONING GOAT BY CROSS-BREED SOMATIC CELL NUCLEAR TRANSFER

Shaneng goat is a famous milking species. Boer goat is world famous goat breed for creophag-ism. In this study, we evaluated the development potential of adult Boer goat' s somatic nuclei after nuclear tansfer(NT) into enucleated MII oocytes of the Shaneng goat. Somatic donor cells were obtained from two different sources: 1)adult granulosa cells(GCs) and 2)adult skin fibroblasts(FCs). The reconstructed embryos that developed to morula or blastocyst stage in vivo were transferred to 38 synchronized recipient. Control: Somatic donor fibroblast cells were obtained from a fetal at 35 day. In the same way the reconstructed embryos were directly transferred to synchronized recipient of Shaneng goats. (1) Experimental group: NT embryos derived from GC and FC developed into morulas and blastocysts at a frequency of 46.8% and 31.4% respectively. Fifty-two NT morula and blastocyst stage embryos were transferred in to 38 recipients, Three of which were confirmed to be pregnant (7.9%). All pregnancies were not maintained to term. (2) Control group: 136 NT embryos were transferred in to 14 recipients, Six of which were confirmed to be pregnant (42. 9% ) . Four of those were maintained to term. Four recipients delivered four male kids(2. 9% of embryos transferred) . One male kid died at birth, the dead lamb shows as "large offspring syndrome", the others appeared health and normal. DNA analysis confirmed that those kids were genetically identical to their donor. These results demonstrated that Shaneng goat somatic cells could direct normal development and Shaneng goat oocyte cytoplasm supported development of preimplantation embryos produced by NT of somatic cell nuclei from Boer goat.