Murine interleukin-18 cDNA cloning and its expression in murine H22 hepatocarcinoma cell

ve To study the expression of murine interleukin-18 (mIL-18) in eucaryotic cells transfected with retroviral vector encoding mIL-18 cDNA, which is the first step towards mIL-18 gene therapy of cancer. Methods Total RNA was extracted from murine hepatic cells and cDNA of mIL-18 was amplified by RT-PCR. The cDNA was introduced into the vector pBluscript and then subcloned into the retroviral vector pLNCX. The pLNCX/mIL-18 vector was transduced into PA317 cells by cationic liposomes and packaged. After selected in medium sup-plemented with 400μg/mL G418, the G418-resistant colonies were isolated and expanded. NIH3T3 and H22 cells were transfected with superna-tant containing pseudotyping retrovirus and underwent selection with G418. The activity of mIL-18 secreted from transfected H22 cells was deter-mined by an enzyme-linked immunosorbent assay (ELISA) of IFN-r production induced by mIL-18. Results The titer of the retroviral superna-tant was 1.5 × 105 CFU/mL. In the presence of 0.15 mg/L Con A, the supernatant collected from conditioned media of the H22 cells transfected with mIL-18 encoding retrovirus enhanced IFN-r production from murine splenocytes. Conclusion We have constructed the recombinant pLNCX/mIL-18 plasmid and the transfected H22 cells expressed mIL-18 in vitro.