Tet-on慢病毒调控系统的构建及其调控作用  

Establishment of Tetracycline-induced gene expression system based on the lentiviral vector

作　　者：韩海勃[1] 赵威[1] 张志谦[1] 

机构地区：[1]北京大学临床肿瘤学院北京肿瘤医院暨北京市肿瘤防治研究所,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142

出　　处：《解剖学报》2011年第6期756-760,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金面上资助项目(30971494;81071733);北京市卫生系统高层次人才培养计划(2011-11-24)

摘　　要：目的构建四环素诱导基因表达的3代慢病毒系统,为慢病毒介导的基因治疗提供实验依据。方法将四环素调控的转录激活因子(rtTA和M2rtTA)分别克隆入带有新霉素(neo)筛选标记的慢病毒载体中,得到pELNS-rtTA-IRES-Neo和pELNS-M2rtTA-IRES-Neo质粒,将四环素顺式应答作用元件(TETO和TREpitt)和绿色荧光蛋白(GFP)克隆入带有杀稻瘟素(blasticidin)筛选标记的慢病毒载体中,得到plenti6-TETO-GFP和plenti6-TREpitt-GFP质粒,将pELNS-rtTA-IRES-Neo与plenti6-TETO-GFP和pELNS-M2rtTA-IRES-Neo与plenti6-TREpitt-GFP分别以10∶1共同转染293细胞,用四环素类似物强力霉素(Dox)诱导GFP基因表达,48h后检测GFP表达。结果成功构建了1代四环素调控体系pELNS-rtTA-IRES-Neo和plenti6-TETO-GFP重组质粒。共转染293细胞后,Dox诱导组的细胞有较强的GFP表达,阳性率约为90%,而未加Dox组细胞GFP阳性表达率约为30%。成功构建了2代四环素调控体系pELNS-M2rtTA-IRES-Neo和plenti6-TREpitt-GFP重组质粒。共转染293细胞后,Dox诱导组的细胞有较强的GFP表达,阳性率约为95%,而未加Dox组细胞未见GFP表达。结论 2代四环素调控体系3代慢病毒系统可以高效诱导目的基因表达,且目的基因背景表达值低。Objective To establish Tetracycline-induced gene expression system for gene therapy based on third generation lentivirus system. Methods The mutant of the reverse Tet transactivator （rtTA and M2rtTA） was subcloned into a lentiviral vector with nee selection marker named as pELNS-rtTA-IRES-Neo and pELNS-M2rtTA-IRES-Neo. The Tetresponsive element （TETO and TREpitt） and green fluorescence protcint （GFP） were subcloned into a leutiviral vector with blasticidin selection marker named as plenti6-TETO-GFP and plenti6-TREpitt-GFP. 293 cells were contransfect with pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP, or with pELNS-M2rtTA-IRES-Neo and plenti6- TREpitt -GFP. Cells were treated by Dox to induce GFP expression. After 48hours, GFP expression in the eo-transfected cells was observed under a fluorescent microscope. Results The first generation of Tetracycline-induced gene expression system named pELNS-rtTA- IRES-Neo and plenti6-TETO-GFP vectors were successfully set up. GFP expression in cotransfected cells induced with Dox was about 90% positive, while there was 30% positive GFP expression observed in no Dox inducing group. The second generation of Tetracycline-induced gene expression system named pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt-GFP vectors were successfully set up. GFP expression in cotransfected ceils induced with Dox was about 95% positive, while there was no positive GFP expression observed in no Dox inducing group. Conclusion Tetracycline-induced gene expression system based on lentivirus was successfully set up, which can induce gene expression effectively and tightly without obvious side-effects on cells by using the second Tetracycline-induced elements.

关 键 词：四环素 慢病毒 基因调控 基因工程 基因克隆 荧光显微术 

分 类 号：Q789[生物学—分子生物学]