AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polyli...AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNA and pMNSIFNB, where the transcription of IFN gene was driven by SV40 early region promoter, and pMNAIFNA, pAMNSIFNA and pMNAIFNB, where the transcription of IFN gene was driven by SV40 early region promoter regulated by α fetoprotein enhancer. The retroviral constructs were respectively introduced into retroviral amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection rate was (4～40)×10 3 colonies/μg DNA/10 6 PA317 cells. The retrovirus infection rate was (5～500)×10 4 colony forming units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells and melanoma cell lines in the presence of 4mg/L polybrene. RESULTS Northern and Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α fetoprotein enhancer induced efficient and apecific transcription and expression of IFNs gene driven by the promoter of different origin in human hepatoma cells by which α fetoprotein was highly produced. CONCLUSION Cis active element of α fetoprotein gene can drive specific expression of IFNs gene in human hepatoma cells, which provides some valuable data for the hepatoma specific immune gene therapy.展开更多
文摘AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNA and pMNSIFNB, where the transcription of IFN gene was driven by SV40 early region promoter, and pMNAIFNA, pAMNSIFNA and pMNAIFNB, where the transcription of IFN gene was driven by SV40 early region promoter regulated by α fetoprotein enhancer. The retroviral constructs were respectively introduced into retroviral amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection rate was (4～40)×10 3 colonies/μg DNA/10 6 PA317 cells. The retrovirus infection rate was (5～500)×10 4 colony forming units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells and melanoma cell lines in the presence of 4mg/L polybrene. RESULTS Northern and Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α fetoprotein enhancer induced efficient and apecific transcription and expression of IFNs gene driven by the promoter of different origin in human hepatoma cells by which α fetoprotein was highly produced. CONCLUSION Cis active element of α fetoprotein gene can drive specific expression of IFNs gene in human hepatoma cells, which provides some valuable data for the hepatoma specific immune gene therapy.
