Figure 5.

Interferon‐stimulated gene responses by transfection of siRNA vectors. (a) Huh7 cells were seeded at 5 × 10⁴ per well in 24‐well plates on the day before transfection. As a positive control, 200 ng of pISRE‐TA‐Luc, or pTA‐Luc, 1 ng of pRL‐CMV, were transfected into a well using FuGENE‐6 Transfection Reagent (Roche), and the cells were cultured with 1 U/mL of interferon (IFN) in the medium (lane 1). Lanes 3–5: 200 ng of pISRE‐TA‐Luc or pTA‐Luc, and 1 ng of pRL‐CMV were cotransfected with (lane2) 300 ng of poly (I : C), or 200 ng of plasmids (lane 3) pcDNA3.1, (lane 4) pUC19‐shRNA‐Control or (lane 5) pUC19‐shRNA‐HCV. Lanes 6–8: 200 ng of pISRE‐TA‐Luc or pTA‐Luc, and 1 ng of pRL‐CMV were transfected, and MOI = 1 of adenoviruses, (lane 6) AxLacZ, which expressed the beta‐galactosidase (LacZ) gene under control of the chicken beta‐actin (CAG) promoter as a control, (lane 7) AxshRNA‐Control or (lane 8) AxshRNA‐HCV were infected. Dual luciferase assays were performed at 48 h after transfection. The Fluc activity of each sample was normalized by the respective Rluc activity, and the respective pTA luciferase activity was subtracted from the pISRE luciferase activity. The experiment was done in triplicate, and the data are displayed as means + SD. (b) Huh7 cells were infected with indicated recombinant adenoviruses, AxLacZ, AxshRNA‐Control and AxshRNA‐HCV. RNA was extracted from each sample at day 6, and mRNA expression levels of an interferon‐inducible MxA protein were quantified by the real‐time RT‐PCR analysis. Primers used were as follows: human MxA sense, 5′‐CGA GGG AGA CAG GAC CAT CG‐3′; human MxA antisense, 5′‐TCT ATC AGG AAG AAC ATT TT‐3′; human beta‐actin sense, 5′‐ACA ATG AAG ATC AAG ATC ATT GCT CCT CCT‐3′; and human beta‐actin antisense, 5′‐TTT GCG GTG GAC GAT GGA GGG GCC GGA CTC‐3′.