体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究

Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer

[目的]探讨供体细胞类型、移植胚胎发育阶段、数量及部位对山羊转基因克隆效率的影响。[方法]利用体细胞核移植技术将转染人乳铁蛋白基因hLF的山羊胎儿成纤维细胞（GFF）和乳腺上皮细胞（GMGE）移植到MII期去核卵母细胞内，经电融合、激活、体外培养后，2-8细胞期克隆胚被移植到同期发情山羊的输卵管内，囊胚被移植到子宫角内。[结果]GFF与GMGE的妊娠率相近（输卵管移植妊娠率分别为26.47%及20.00%）；在GFF，输卵管移植的妊娠率与子宫角内移植妊娠率接近（分别为26.47%及25.00%），输卵管移植胚胎平均数为21.2组的妊娠率显著高于5.93组和9.64组（40.00%及26.67%，21.43%）。[结论]供体细胞类型、移植胚胎的发育阶段及移植部位对山羊转基因克隆效率的影响不大，但对于输卵管移植，受体羊移植胚胎数量对妊娠率有明显的影响。此外，该研究还提示了利用成年羊乳腺上皮细胞制作转基因动物的可行性。

[ Objective ] The aim of this study is to understand the effects of donor cell type, embryo stage, number and transfer position on the efficiency of goat transgenic clone. [ Method] Using somatic cell nuclear transfer technology, the single goat fetal fibroblasts （GFF） and mammary gland epithelial cells （GMGE） harboring human lactoferrin （hLF） gene were transferred to the enucleated oocyte. Reconstructed karyoplastcytoplast couplets were fused, activated, and cultured in vitro. Embryos at 2 -8 cell stage were transferred into oviduct of synchronized recipients, and blastocysts were transferred into uterine horn. [ Result] The pregnancy rate was similar between GFF and GMGE （oviduct transfer： 26.47% vs. 20.00% ）, and between oviduct transfer and uterine horn transfer （26.47% vs. 25.00% ） for GFF group; pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group （40.00% vs. 26.67% and 21.43% ）. [ Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldnt be affected by donor cell type, embryo stage and transfer position but he done by the number of embryo transferred per recipient. In addition, the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.