由成年转基因山羊体细胞而来的克隆山羊

在已经获得的乳腺特异性表达人促红细胞生成素 (rhEPO)成年转基因山羊 (Caprahircus)的基础上 ,取其耳尖成纤维细胞和卵巢颗粒细胞 ,进行体外传代培养 ,然后将这种培养的转基因山羊的体细胞移入去核的处于第Ⅱ次减数分裂中期的卵母细胞中 ,并进行电融合 ,构建重构胚胎 ,重构胚胎在体内培养 6d ,再将发育至囊胚或桑椹胚的重构胚胎移入同步情期的寄母羊子宫内。结果 ,有 2只寄母羊妊娠并最终产下 2只成活的克隆山羊。她们分别来自同一成年母羊的耳尖成纤维细胞和卵巢颗粒细胞。克隆羊经PCR RFLP图谱分析显示 :以克隆羊组织DNA为模板的PCR产物与相应的提供体细胞的基因羊的PCR产物的酶切图谱完全一致 ;并且经PCR对外源hEPO基因检测表明 2只克隆山羊均携带hEPO外源基因。由此证明获得了转基因成年体细胞的克隆山羊

Cloned Goats Produced from the Somatic Cells of an Adult Transgenic Goat

This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.