论文部分内容阅读
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 construct tetracycline-inducible gene of third generation lentivirus system and provide experimental evidence for lentivirus-mediated gene therapy. METHODS: Tetracycline-regulated transcriptional activators (rtTA and M2rtTA) were cloned into lentiviral vector with neo selection marker to obtain pELNS-rtTA-IRES-Neo and pELNS-M2rtTA-IRES-Neo plasmids, The tetracycline cis-responsive elements (TETO and TREpitt) and green fluorescent protein (GFP) were cloned into lentiviral vectors with the blasticidin selection marker to yield plenti6-TETO-GFP and plenti6-TREpitt-GFP 293 cells were co-transfected with pELNS-rtTA-IRES-Neo with plenti6-TETO-GFP and pELNS-M2rtTA-IRES-Neo with plenti6-TREpitt- GFP, respectively, at 10: 1 with a tetracycline analogue of doxorubicin ) Induced GFP gene expression 48h after the detection of GFP expression. Results The recombinant plasmid pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP were successfully constructed. Cotransfected 293 cells, Dox-induced cells have a strong GFP expression, the positive rate of about 90%, while the cells without Dox group GFP-positive expression rate of about 30%. The 2nd generation tetracycline regulatory system pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt-GFP recombinant plasmids were successfully constructed. Cotransfected 293 cells, Dox-induced cells have strong GFP expression, the positive rate of about 95%, while no Dox group cells were not GFP expression. Conclusion Generation 2 tetracycline regulatory system 3 lentiviral system can efficiently induce the target gene expression, and the purpose of gene expression is low.