绿色荧光小鼠胚胎干细胞的分离与培养

The isolation and cultivation of the EGFP mouse embryo stem cell

目的:建立绿色荧光小鼠胚胎干细胞系。方法:以ICR及GFP转基因小鼠为实验动物,冲取交配后3.5天小鼠GFP-/+囊胚,去透明带后进行完全培养,以13.5天鼠胚胎成纤维细胞(MEF)为饲养层,并对细胞克隆用亚克隆的方法分离扩大培养。结果:获得了边缘清晰,表面平滑,结构致密,隆起生长的鸟巢状克隆,碱性磷酸酶染色呈强阳性,干细胞特异性的多能因子OCT4及Nanog表达呈阳性,且能稳定表达绿色荧光的雄性干细胞系。结论:本文为小鼠ES细胞系的建立提供了一种稳定而可靠的方法。

Objective：To establish a EGFP mouse ES cell line.Methods：Blastocysts which were from the female ICR mouses mated with the male GFP transgenic mouse（C57BL/6） were collected,and the zone pellucida of each blastocyst was removed by incubating in pronase solution,then the blastocysts were plated onto feeder cells of mitotically mouse embryo fibroblasts（MEFs）.Result：after culturing and passaging,a cell line was got which was eage clear,surface smooth,structure compact,and nest-like,these cells showed positive alkaline phosphatase activity,expressed primate ES-special markers OCT4 and Nanog,and expressed EGFP stably.Conclusion This article provides a simple,stable and reliable method for the establishment of mouse ES cell line.