Construction of retroviral vectors to induce a strong expression of human class interferon gene in human hepatocellular carcinoma cells in vitro

摘要

AIM: To establish the hepatoma cell–specific expression of human interferon (IFN) gene mediated by retroviral vectorsMETHODS: Human interferon α and interferon β complementary DNA (IFN cDNA) were cloned into the polylinker site of pMNSM retroviral vector to construct recombinant retroviral vectors pMNSIFNA and pMNSIFNB, with the transcription of IFN gene being driven by Simian virus 40 early region promoter (SV40) early region promoter. IFN cDNAs were also cloned into pMNAIFNA, pAMNSIFNA, and pMNAIFNB, with the transcription of IFN gene being driven by SV40 early region promoter regulated by α-fetoprotein enhancer. Next, the retroviral constructs were introduced into retroviral amphotropic packaging cells using the lipofectamine-mediated gene transfer procedure. The rate of plasmid transfection was (4-40) × 10³ colonies/μg DNA/10⁶ PA317 cells. The rate of retrovirus infection was (5-500) × 10⁴ colony forming units (CFU)/mL. Further, the recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells, and melanoma cell lines in the presence of 4 μmg/L polybrene.RESULTS: Northern and Dot hybridization of total RNA from the neomycin-resistant colonies and IFN expression assay indicated that human α fetoprotein enhancer induced efficient and specific transcription and expression of IFN genes driven by the promoter of different origins in human hepatoma cells, leading to high production of α fetoprotein.CONCLUSION: Cis active element of α-fetoprotein gene can drive specific expression of IFN genes in human hepatoma cells, which provides some valuable data for the hepatoma-specific immune gene therapy.