Nanog慢病毒载体构建及在胚胎干细胞分化调控中的应用  

Construction of Nanog lentiviral expression vector and its application in differentiation regulation of embryonic stem cells

作　　者：陈霏[1] 万利[2] 李艳[3] 李欣[4] 

机构地区：[1]广州医科大学基础学院组织学与胚胎学教研室,广东省广州市511436 [2]广州医科大学附属第四医院检验科,广东省广州市511447 [3]广州医科大学附属乐从医院,广东省佛山市528315 [4]广州医科大学药学院药理学系,广东省广州市511436

出　　处：《中国组织工程研究》2016年第19期2763-2769,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年基金(31401240);广东省医学科研基金(B2014190;B2014185);广州医科大学科研项目(2013C01;L135060)~~

摘　　要：背景:在胚胎干细胞分化调控研究中,仍缺少安全有效的组织特异性启动子介导的荧光报告基因的慢病毒表达载体。目的:构建一个表达多能干细胞组织特异性启动子Nanog同时携带荧光报告基因和抗生素筛选基因的慢病毒表达载体Puro-Nanog-hr GFP,建立小鼠胚胎干细胞转基因细胞系,并观察其在小鼠胚胎干细胞细胞诱导分化过程中的表达。方法:利用PCR扩增Nanog基因,通过LR反应连接荧光报告基因hr GFP构建慢病毒表达载体Puro-Nanog-hr GFP。慢病毒包装并转导小鼠胚胎干细胞后运用嘌呤霉素筛选得到小鼠胚胎干细胞转基因细胞系并进行鉴定,然后诱导其分化,观察随着小鼠胚胎干细胞的分化Nanog的表达情况。结果与结论:通过特定引物PCR鉴定证实,慢病毒表达载体Nanog-hr GFP构建成功,将其成功导入小鼠胚胎干细胞,经筛纯化得到表达绿色荧光的小鼠胚胎干细胞单克隆细胞系,并通过诱导其分化可观察到绿色荧光表达逐渐减弱,可为进一步调控干细胞分化奠定基础。BACKGROUND： There is a lack of safe and effective modular lentivectors in differentiation regulation of embryonic stem cells. OBJECTIVE： To construct a modular lentivector, Puro-Nanog-hr GFP, with Nanog promoter-controlled humanized renilla reniformis green fluorescent protein（hr GFP） and puromycin and to observe the expression of Nanog during the differentiation of Nanog-hr GFP-transfected mouse embryonic stem cell lines. METHODS： After PCR amplification, Nanog, hr GFP and entry vector were recombined into the p Dest-puro vectors to generate the lentiviral expression vector, Puro-Nanog-hr GFP, by the LR reaction. Through lentivirus production, transduction and puromycin screening, the transduced cell lines with Nanog-hr GFP gene were generated and identified. Expression of Nanog during the differentiation of transgenic mouse cell lines was detected. RESULTS AND CONCLUSION： The lentiviral expression vectors Puro-Nanog-hr GFP were constructed successfully by Gateway technology, and then the transducted cell lines were obtained by lentiviral infection. The expression of Nanog was gradually decreased during the process of transgenic cell lines differentiation, which provides a new tool for further investigation on regulation of stem cell differentiation.

关 键 词：干细胞 胚胎干细胞 转基因 组织工程 慢病毒 GATEWAY技术 NANOG 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]