Study On Regulated Retroviral Vectors Containing The Apoptin Gene In The Induction Of Apoptosis In Human Heptoma Cell Line

Objective To construct a tetracycline-regulated recombinant retroviral vector carrying apoptin gene and investigate the effect of apoptin gene expression on HepG2, a human heptoma cell line in the control of tetracycline.Methods The apoptin gene was cloned into a retroviral vector pRevTRE to obtain the recombinant vector pRevTRE-VP3. pRevTRE-VP3 and pRevTet-Off were transfected into PT67 respectively. After antibiotic selection and determining the viral titers, the cell lines PT67/Off and PT67/VP3 that can stably produce virus were selected. The supernate of PT67/Off and PT67/VP3 containing retroviral vectors was collected and was, in turn, used to infect HepG2 cells. After antibiotic selection, the resulting cell lines HepG2/Off-VP3 were established. Apoptin gene expression in HepG2/Off-VP3 cells was controlled by the presence or the absence of tetracycline in medium. After Annexin V-FITC/PI staining, the apoptosis rate in HepG2/Off-VP3 cells was analysed with flow cytometry.Results pRevTRE-VP3 was identified by restrictive enzyme digestion and sequencing. PT67/Off and PT67/VP3, the stable virus-producing package cell lines were obtained and the viral titers were 1×104cfu/ml and 3×104cfu/ml respectively. A heptoma cell line HepG2/Off-VP3-8 that can express apoptin in the control of tetracycline was constructed. The integration and expression of apoptin gene in HepG2/Off-VP3-8 was confirmed by PCR and RT-PCR. HepG2/Off-VP3-8 cells were incubated in medium free of tetracycline or in medium with 1μg/ml tetracycline for 24 hours, 48 hours and 72 hours. The apoptosis rate in the HepG2/Off-VP3-8 cells incubated in medium free of tetracycline was significant higher than that in the HepG2/Off-VP3-8 cells incubated in medium with 1 μg/ml tetracycline at the same time point.Conclusion 1. The tetracycline-regulated retroviral vector carrying apoptin gene is constructed. 2. After apoptin gene is transduced into HepG2 with RevTet system, it can express apoptin and induce apoptosis in HepG2 cells in the control of tetracycline.