高效建立129S1/SvImJ小鼠胚胎干细胞方法的探讨

Research on the high efficient methodology of Establishment of 129S1/SvImJ murine embryonic stem cells.

选取超排后129S1/SvImJ品系小鼠的优质囊胚,使用辐照后冻存复苏的小鼠胚胎成纤维细胞作为饲养层,对比高浓度酶短时间消化法和低浓度酶长时间消化法,以0.25%胰酶-0.04%EDTA高浓度酶短时间消化法高效建立了4株小鼠胚胎干细胞系,建系率为36.3%.碱性磷酸酶,核型分析,体外分化和体内分化能力以及小鼠胚胎干细胞表面特异性分子标志0ct-4和Nanog的检测等表明,4个ES细胞系均为典型的小鼠胚胎干细胞系.所建立的ES细胞系为大规模培养ES细胞,进行后续的实验建立了理想的材料平台.

The high quality embryos of 129S1/SvImJ strain mouse were chosen after superovulation, and directly seeded in the irradiated frozen-thaw mouse embryonic fibroblast cells. Compared with the low concentration long time digestion, the high concentration short time digestion was adopted in the study, and 4 lines of mouse embryonic stem cells were successfully established by trypsinization of 0.25% trypsin-0.04% EDTA, which is the high concen tration short time digestion method. The ratio of establishment cell lines is 36.3%, and the cell strains exhibited the typical ES cell characteristic identified by different ways including the AKP and karyotype assay, the differentiation potential in vivo and in vitro, and the examination of specific surface markers expression of Oct-4 and Nanog. The ES cell lines can be made scalable culture and used as an ideal experimental material for the further research.