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

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.