CREG基因敲除小鼠胚胎干细胞的筛选及鉴定  

Selection and Identification of Mouse Embryonic Stem Cells with CREG Gene Knock Out

作　　者：田孝祥[1] 张剑[1] 陶杰[1] 孙鸣宇[1] 闫承慧[1] 韩雅玲[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科,辽宁沈阳110840

出　　处：《现代生物医学进展》2012年第32期6221-6224,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金重点项目(81130072);国家自然科学基金面上项目(81070097);国家科技部973前期项目(2011CB512111);辽宁省科技攻关项目(2010225036)

摘　　要：目的:为阐明E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)在发育过程中的作用,本研究拟通过高浓度药物筛选获得基因敲除的小鼠胚胎干细胞(Embryonic stem cell,ESC)。方法:用0.5 mg/ml、1.0 mg/ml、1.5 mg/ml、2.0mg/ml、2.5 mg/ml及3.0 mg/ml 6个浓度的G418培养CREG杂合型(CREG其中一个等位基因被新霉素抗性neo基因替代)小鼠ESC 2周,确定最佳的G418筛选浓度。挑取该浓度下存活的ESC克隆进行扩增。将每个ESC克隆一半冻存,另一半贴壁培养。待ESC生长至80%融合后分别提取基因组DNA和蛋白。PCR方法扩增CREG基因明确基因组中是否存在CREG基因,WesternBlot方法鉴定是否有CREG蛋白表达。结果:确定2.0 mg/ml G418为最佳的筛选浓度。在该浓度下,共获得存活的克隆10个,PCR证实C2及C7克隆基因组中没有CREG基因,Western Blot证实C2及C7无CREG蛋白表达。结论:成功获得CREG基因敲除的小鼠ESC 2株,为深入研究CREG功能奠定了基础。Objective： To clarify function of CREG （Cellular repressor of E1A-stimulated genes） during differentiation, we aimed to obtain CREG gene knock out mouse ESC （Embryonic stem cell） by drug selection at high concentration. Methods： Mouse heterozygous CREG ESC （one allele of CREG gene was targeted by neomycin resistant neo gene） was cultured trader six concentrations of G418, i.e. 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 2.5 mg/ml and 3.0 mg/ml for 2 weeks. An appropriate selection conccntration was determined and ESC colonies survived under this concentration were picked up and expanded. For each selected ESC clone, one half was cryopre-served and the other half was cultured until reaching 80 % confluence. Genomic DNA and protein were extracted from the confluent ESC. PCR method was applied to detect the existence of genomic CREG gene and Western blot were used to determine CREG protein expression. Results： The 2.0 mg/ml was determined to be the appropriated G418 concentration. Under this concentration, 10 survived ESC clones were obtained. PCR results identified that clone C2 and C7 ESC had no CREG gene in genome, and Western blot analysis showed that there was no CREG protein expression in clone C2 and C7. Conclusion： We successfully obtained 2 clones of CREG knocked out mouse ESC, which will pave the way for the further investigation on CREG function.

关 键 词：CREG 基因敲除 胚胎干细胞 

分 类 号：Q291[生物学—细胞生物学]