稳定过表达Olig2的小鼠胚胎干细胞系的建立  

Establishment of stably Olig2-overexpressed mouse embryonic stem cells

作　　者：王贝[1] 罗梦娇[1] 郭瑞[1] 董富兴[1] 陈彦[1] 王会平[1] 曲学彬[1] 姚瑞芹[1] 

机构地区：[1]徐州医学院生物学教研室,徐州221009

出　　处：《神经解剖学杂志》2014年第4期425-430,共6页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81271345;81302519);江苏省自然科学基金(BK20131132;BK20130221)

摘　　要：目的:建立稳定过表达Olig2的小鼠胚胎干细胞(mESCs)系。方法:从含有Olig2的质粒中利用PCR技术克隆Olig2基因编码序列,酶切连接入GV218载体,形成Olig2-GV218重组载体。将构建好的Olig2-GV218载体与两种病毒辅助包装原件载体质粒共转染293T细胞,制备并浓缩慢病毒颗粒,侵染mESCs,嘌呤霉素筛选的阳性克隆传代培养10代后鉴定细胞内Olig2的表达。结果:PCR扩增得到含Olig2基因编码序列的特异性条带。重组的Olig2-GV218载体中Olig2编码序列与目标序列几乎一致。携带有Olig2-GV218载体的慢病毒能够正常感染mESCs,表达Olig2-GFP融合蛋白,并稳定遗传。结论:成功建立了稳定过表达Olig2的小鼠胚胎干细胞系。Objective： To establish stably Olig2-overexpressed mouse embryonic stem cells （mESCs）. Methods： Olig2 coding sequences, which were cloned from vector containing Olig2 cDNA by PCR, were digested and ligated into vector GV218 to form Oligy2-GV218 recombinants. The recombined Olig2-GV218 vectors, together with other two Lentiviral helper vectors, were co-transfected into 293T cells to product and concentrate Lentiviral particles. After infected with Olig2.- GV218 Lentiviral, the puromycin resistant mESCs were cultured for 10 passages, and then the expression of Olig2 was tested. Results： The Olig2 coding sequences were successfully cloned by PCR, and the cloned Olig2 sequences were almost the same with subject. The mESCs, which were infected by Olig2-GV218 Lentiviral efficiently, showed a genetic stability of Olig2-GFP fusion protein expression. Conclusion： The stably Olig2-overcxpressed mESCs were successfully established.

关 键 词：小鼠胚胎干细胞 OLIG2 过表达 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]