不同类型的转基因细胞核供体对生产小鼠转基因克隆胚胎的影响

目的 利用体细胞核移植(SCNT)技术生产小鼠转基因克隆胚胎. 方法 利用所构建的含新霉素抗性(Neor)基因和增强型绿色荧光蛋白(EGFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染小鼠胎儿成纤维细胞和小鼠胚胎干细胞,经G418筛选14d后用于核移植.同时以未转基因小鼠卵丘细胞和成纤维细胞为核供体,进行体细胞核移植. 结果 转基因与未转基因胎鼠成纤维细胞的重构胚的囊胚(154/160)发育率为19.23%和22.91%,差异不显著(P＞0.05);转基因胚胎干细胞与胎鼠成纤维细胞的重构胚的囊胚(152/154)发育率为41.54%和19.23%,差异显著(P＜0.05);未转基因卵丘细胞与成纤维细胞的重构胚的囊胚(171/160)发育率为41.17%和22.91%,差异显著(P＜0.05). 结论 利用体细胞核移植技术,小鼠胎儿成纤维细胞和胚胎干细胞可以有效地生产小鼠转基因囊胚.

Production of transgenic cloned mouse embryos by somatic cell nuclear transfer with different donor cells

Objective To generate transgenic mouse embryos by somatic cell nuclear transfer (SCNT), and compare effects of different donor cells on developmental potential of cloned embryos. Methods Mouse embryo fibroblasts and mouse embryonic stem (ES) cells were transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (GFP) and neomycin resistant (Neor) genes by electroporation. Transgenic cell lines were obtained for SCNT after 14 days selection with G418. At the same time, SCNT was performed using cumulus cells and normal fibroblasts as control. Results There was no significant difference in blastocyst rates between reconstructed embryos derived from transgenic fibroblasts and nontransgenic ones (154/160,19.23% vs 22.91%, EM>P/EM>＞0.05). Reconstructed embryos from transgenic ES cells showed higher blastocyst developmental rates than that from transgenic fibroblasts donor (152/154,41.54% vs 19.23%, P<0.05). Cumulus cloned embryos had better developmental potential than that of fibroblasts cloned ones (171/160, 41.17% vs 22.91%, P<0.05). Conclusion Our results showed that transgenic donor cells did not affect EM>in vitro/EM> developmental potential of mouse SCNT embryos. Using EGFP maker, mouse transgenic blastocysts could be produced effectively