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.

作　　者：姜祖韵[1] 吴蓉蓉[1] 袁毅君[2] 陈良标[3] 赵小立[1] 陆永良[4] 姚行[4] 戴利成[4] 张铭[1] 

机构地区：[1]浙江大学生命科学学院,浙江杭州310012 [2]天水师范学院生命科学与化学学院,甘肃天水741000 [3]中国科学院遗传与发育生物学研究所,北京100101 [4]湖州市中心医院,浙江湖州313000

出　　处：《浙江大学学报（理学版）》2005年第5期574-578,共5页Journal of Zhejiang University（Science Edition）

基　　金：浙江省重大资助项目(J20020579-30116);湖州中心医院合作项目(H20010984-32536)

摘　　要：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.

关 键 词：129S1/SvImJ小鼠 囊胚 饲养层 消化方法 胚胎干细胞 

分 类 号：Q813.11[生物学—生物工程]