过表达Olig2和Nkx2.2人胚胎干细胞系的建立  

Establishment of Olig2 and Nkx2.2 overexpressed human embryonic stem cells

作　　者：杨丽华[1,2] 王婷[2] 马丽[2] 罗梦娇[2] 曲学彬[2] 武秀香[2] 姚瑞芹[2] 

机构地区：[1]第二军医大学细胞生物学教研室,上海200433 [2]徐州医学院细胞生物学与神经生物学教研室,徐州221009

出　　处：《解剖学杂志》2015年第6期645-648,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金(81271345,81302515);江苏省自然科学基金(BK20131132,BK20130221);徐州医学院院课题(2012KJ07);2014年度“青蓝工程”中青年学术带头人培养项目

摘　　要：目的:建立过表达Olig2和Nkx2.2的人胚胎干细胞(hESCs)系.方法:利用RT-PCR从鼠N2A细胞克隆出Olig2和Nkx2.2的ORF片段,将Olig2和Nkx2.2基因编码序列分别和带有HA及Flag的载体连接,构建Olig2-HA和Nkx2.2-Flag重组载体.将构建的载体分别与2种病毒辅助包装原件载体质粒共转染293T细胞,制备并浓缩Olig2-HA和Nkx2.2-Flag慢病毒颗粒,感染hESCs,嘌呤霉素筛选的阳性克隆传代培养10代后鉴定细胞内Olig2和Nkx2.2的表达.结果:PCR扩增得到含Olig2和Nkx2.2基因编码序列的特异性条带;携带有Olig2和Nkx2.2基因的慢病毒能够同时感染hESCs,表达Olig2-HA和Nkx2.2-Flag融合蛋白,并稳定遗传.结论:成功构建了同时过表达Olig2和Nkx2.2的人胚胎干细胞系.Objective:To establish Olig2 and Nkx2.2 overexpressed human embryonic stem cells(hESCs).Methods-,The complete open reading frames(ORFs) of 01ig2 and Nkx2.2 were cloned from mouse N2 A cell by reverse transcription polymerase chain reaction(RT-PCR).OIig2 and Nkx2.2 gene coding sequences were respectively ligated to the vectors with HA and Flag tag to construct recombinants Olig2-HA and Nkx2.2-Flag.The two recombinants were separately cotransfected with other two lentiviral helper vectors into 293 T cells,and lentiviral particles were produced and concentrated,which could infect hESCs and make the puromycin resistant hESCs cultured for 10 passages,and then the expressions of Olig2 and Nkx2.2 were identified.Results:Olig2 and Nkx2.2 gene coding sequences were successfully amplified by PCR.The hESCs,which could be simultaneously infected with Olig2 and Nkx2.2 lentivirals,showed a genetic stability of Olig2-HA and Nkx2.2-Flag fusion proteins expression.Conclusion:Olig2 and Nkx2.2 overexpressed hESCs are successfully established.

关 键 词：OLIG2 Nkx2.2 过表达 人胚胎干细胞 克隆 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]