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. 1997 Sep 15;3(3):139-42.
doi: 10.3748/wjg.v3.i3.139.

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

G W Cao  1 , Jun-Gao  1 , P Du  1 , Z T Qi  1 , X T Kong  1
Affiliations

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

G W Cao et al. World J Gastroenterol. .

Abstract

Aim: To establish the hepatoma cell-specific expression of human interferon (IFN) gene mediated by retroviral vectors

Methods: 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) × 10(3) colonies/μg DNA/10(6) PA317 cells. The rate of retrovirus infection was (5-500) × 10(4) 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.

Keywords: Carcinoma, hepatocellular; Gene expression; Gene therapy; Genes, regulatory; Interferon alpha; Interferon beta; Liver neoplasms; Retroviridae.

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Figures

Figure 1
Figure 1
Structure of MNSM, MNSIFNB, MNAIFNB, MNSIFNA, MNAIFNA, and AMNSIFNA retroviral vectors. LTR: Retroviral long-terminal repeat sequence; Neo R: Neomycin phosphotransferase gene for G418 selection; SV40: Simian virus 40 early region promoter; HuIFN-β-cDNA: Human interferon β complementary DNA; HuIFN-α-cDNA, Human interferon α complementary DNA. AFPe: Human α-fetoprotein “house-keeping gene” enh ancer; AFPe.c, Human α-fetoprotein “house-keeping gene” enhancer core sequence.
Figure 2
Figure 2
Northern blot of total RNA from the tumor cells modified with human interferon-α gene, probed with 32P labeled human interferon-α cDNA. Lane 1: Unmodified HepG2 cells; Lane 2: MNSM modified HepG2 cells; Lane 3: MNSIFNA modified HepG2 cells; Lane 4: MNAIFNA modified HepG2 cells; Lane 5: MNSIFNB modified HepG2 cells; Lane 6: MNSIFNA modified HepG2 cells; Lane 7: AMNSIFNA modified HepG2 cells; Lane 8: MNAIFNB modified HepG2 cells.
Figure 3
Figure 3
Dot blot of total RNA from human tumor cells modified with HuIFN-β gene, and fully selected with G418, probed with 32P labeled HuIFN-β-cDNA. Lane 1: Hep3B cells infected with MNAIFNB retroviruses; Lane 2: Hep3B cells infected with MNSIFNB retroviruses; Lane 3: HepG2 cells infected with MNAIFNB retroviruses; Lane 4: HepG2 cells infected with MNSIFNB retroviruses; Lane 5: M21 cells infected with MNSIFNB retroviruses.
See this image and copyright information in PMC

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