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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。
Objective To produce mouse transgenic cloned embryos using somatic cell nuclear transfer (SCNT) technology. Methods The double-labeled selection vector containing Neor gene and enhanced green fluorescent protein (EGFP) gene was constructed and transfected into mouse fetal fibroblasts and mouse embryos by electroporation Stem cells, after G418 screening 14d for nuclear transfer. At the same time, non-transgenic mice cumulus cells and fibroblasts as nuclear donors, somatic cell nuclear transfer. Results The developmental rates of blastocysts (154/160) in reconstructed embryos of transgenic and non-transgenic fetal rat fibroblasts were 19.23% and 22.91%, respectively, with no significant difference (P> 0.05). Transgenic embryonic stem cells and fetal rat fibroblasts The blastocysts (152/154) of the reconstructed embryos developed 41.54% and 19.23%, respectively (P <0.05). The blastocysts (171/160) of the reconstructed embryos of non-transgenic cumulus cells and fibroblasts developed Rates were 41.17% and 22.91%, the difference was significant (P <0.05). Conclusion The transgenic blastocysts can be efficiently produced in mouse embryonic fibroblasts and embryonic stem cells by somatic cell nuclear transfer.