Gene Apoptin Transducted by Retroviral Vectors in Induction of Apoptosis in Human Colonic Carcinoma Cell Line Lovo

Objective To construct a doxicyclin-induced recombinant retroviral vector carrying apoptin gene and investigate the effect of apoptin gene expression on Lovo, a human colon cancer cell line after induced by doxicyclin. Methods The apoptin gene was cloned into retroviral vector pRevTRE to obtain the recombinant vector pRevTRE-VP3. pRevTRE-VP3 and pRevTet-on were transfected into PT67 respectively. Retroviral vectors were collected and were, in turn, used to infect Lovo cells. After antibiotic selection, the resulting cell lines Lovo/on-vp3 was established. The expression of apoptin in Lovo/on-vp3 cells was controlled by the presence or the absence of doxicyclin in medium. After AnnexinⅤ-FITC/PI staining, the apoptosis rate of Lovo/on-vp3 cells was analyzed by flow cytometry. Results After restriction enzyme digestion and sequencing,recombinant retroural vector pRevTRE-VP3 was successfully constructed and added a kozak sequence,selected via tetracycline-controlled gene expression in apoptosis of human colon cancer cell line Lovo. By PCR and RT-PCR confirmed that apoptin gene has been integrated into the cellular DNA; and apoptosis in the culture environment to foster doxycycline in the dosage of 1mg/L was significantly higher than those without doxycycline at the same points of 24h,48h and 72h.The difference was significant. Conclusion Gene vp3 was introduced into human colonic carcinoma cell Lovo via RevTet system, can express protein apoptin and lead apoptosis after induced by doxicyclin. The expression of protein apoptin and the rate of apoptosis increases as extends the induce time.