Retroviral gene transfer into liver requires efficient virus uptake and proviral integration. To address retroviral receptor activity in vivo, we developed a novel human liver cell line as convenient models are lacking. The mouse ecotropic retrovirus receptor cDNA (Closs et al., J. Biol. Chem.,1993;268:7538) was cloned into the CMV expression cassette of the plasmid pCDNA3 (Invitrogen Corp.), which coexpresses neor gene. After gene transfer via liposomes into Huh-7 cells, G418-resistant Huh-7.MCAT-l cells were selected and dilutionally cloned. In one clone, Huh-7.MCAT-1.7, RNA transblotting showed a unique transcript hybridizing with MCAT-1 cDNA, not observed in parental Huh-7 cells. The Huh-7.MCAT-1 cell clones showed no change in morphology or proliferation determined by [3H]-thymidine incorporation and cell counts. To demonstrate MCAT-1 receptor activity, we assayed its cationic amino acid transporter function using L-arginine. In response to a 2 hr [3H]-arginine pulse, Huh-7.MCAT-1.7 cells exhibited markedly increased arginine uptake compared with Huh-7 cells (7±2 fold increase, p<0.001) or NIH 3T3 mouse fibroblasts (3+1 fold increase, p<0.01). The arginine uptake was increased in Huh-7.MCAT-I.7 cells by dexamethasone and further increased by dexamethasone, insulin and hepatocyte growth factor in combination, p<0.005. To demonstrate specific activity of the ecotropic receptor, we reasoned that Huh-7.MCAT-1 cells would be susceptible to ecotropic retroviruses, whereas Huh-7 cells are not. When cells were exposed at MOI (multiplicity of infection) of 2 to an ecotropic LacZ retrovirus infecting 93%±4%NIH3T3 cells, 19%±4% Huh-7.MCAT-1.7 cells but only 0.7%±0.8% Huh-7 cells turned blue on X-gal staining, p<0.001. In 20 other Huh-7.MCAT-1 clones derived by single cell cloning, the retroviral transduction efficiency (virus MOI 2) was also increased, ranging from 7%±4% to 32%±4%. After exposure to more ecotropic virus, MOI 25, 65%±5% Huh-7.MCAT-1.7 cells stained blue, confirming a dose-response activity of the MCAT-1 receptor. As Huh-7.MCAT-1 cells express physiologically regulatable ecotropic retroviral receptors, this will facilitate studies of retroviral receptor regulation and gene therapy.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)