张鹏,杨红兰,刘含,周艳华,杨燕,范安然,何志旭,舒莉萍
ZHANG Peng,YANG Honglan,LIU Han,ZHOU Yanhua,YANG Yan,FAN Anran,HE Zhixu,SHU Liping

• 1:贵州医科大学细胞工程生物医药技术国家地方联合工程实验室组织工程与干细胞实验中心贵州省再生医学重点实验室
• 2:中国医学科学院成体干细胞转化研究重点实验室
• 3:遵义医科大学附属医院儿科学教研室

摘要(Abstract)：

目的:研究甲基结合蛋白1(Mbd1)对小鼠胚胎干细胞克隆形态及增殖的影响。方法:设计针对Mbd1转录起始位点的gRNA,将表达有gRNA和Cas9蛋白的质粒使用脂质体转染方法转入小鼠胚胎干细胞(J1);使用抗生素将未转染成功的细胞杀死后,细胞继续培养7 d,挑取单克隆细胞,采用PCR法筛选敲除Mbd1的纯合子细胞,观察Mbd1缺失对小鼠胚胎干细胞克隆形态的影响;使用细胞计数法检验Mbd1对胚胎干细胞增殖的影响。结果:通过Cripsr/Cas9成功获得敲除Mbd1的胚胎干细胞,Mbd1缺失后的小鼠胚胎干细胞丧失了常规小鼠胚胎干细胞的3D克隆形态,细胞呈现单层扁平克隆;Mbd1缺失后小鼠胚胎干细胞增殖速率变慢。结论:Mbd1通过影响小鼠胚胎干细胞克隆形态和增殖而影响胚胎干细胞的多能性。
Objective: To investigate the effect of methyl-CpG binding domain protein 1(Mbd1) on the colony morphology and proliferation of mouse embryonic stem cells(mESCs). Methods: A gRNA was designed to specifically target to the transcriptional start site of Mbd1 and cloned into pX459 plasmid, and then co-transfected with the plasmid which contains Cas9 gene into mESCs. mESCs expressing gRNA were selected by puromycin treatment, cultured for 7 days to form a colony and then the Mbd1 deletion was verified by genotyping using PCR. The efficiency of Mbd1 deletion was further confirmed using Western Blot. The morphology of mESCs was observed and cell proliferation was measured using cell counting method. Results: The expression of Mbd1 was completely abrogated by Cripsr/Cas9. The 3 D-colony morphology of mESCs was lost in Mbd1 knockout mESCs. The proliferation of Mbd1 knockout cells was slower than the wildtype cells. Conclusion: Mbd1 is important for the pluripotency maintenance of mESCs via regulating the colony formation and cell proliferation.

关键词(KeyWords)： 甲基结合蛋白;胚胎干细胞;多能性;细胞增殖;神经前体细胞;基因敲除
methyl-CpG binding domain protein;embryonic stem cell;pluripotency;cell proliferation;neural precursor cells;gene knckout

Abstract:

Keywords:

基金项目(Foundation): 贵州医科大学博士启动基金[院博合J字(2018)016号];; 国家自然科学基金项目(31660316);; 中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助(2017PT31042);; 贵州省教育厅导师工作室[黔教研合GZS(2016)02]

作者(Author): 张鹏,杨红兰,刘含,周艳华,杨燕,范安然,何志旭,舒莉萍
ZHANG Peng,YANG Honglan,LIU Han,ZHOU Yanhua,YANG Yan,FAN Anran,HE Zhixu,SHU Liping

DOI: 10.19367/j.cnki.1000-2707.2019.06.001

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