FIGURE 1.

Down-regulation of Aspm gene in mouse hepatocytes expressing HCV NS5A protein. A, liver-specific expression of NS5A protein driven by the transthyretin promoter. HepG2, Huh7, A549, and COS7 cells were transfected with 15 μg of plasmid pAdTrack-CMV-T7HisNS5A or pAdTrack-TTRB-T7HisNS5A as indicated. Forty-eight hours post-transfection, the cells were harvested, and cell lysates were subjected to Western blot analysis with antibodies specific to the viral NS5A protein, GFP, and the control GAPDH. B, expression of GFP protein in the tissue sections of hydrodynamic-injected mouse. Plasmid pAdTrack-TTRB-T7HisNA5A that encodes GFP and NS5A protein was injected into mouse tail vein by hydrodynamics-based method. Two days post-injection, mouse tissue sections from various origins as indicated were prepared. Expression of GFP in the tissue sections was monitored by a fluorescence detector. C, co-expression of HCV NS5A protein in the GFP-expressing mouse hepatocytes. Hepatocytes from three mice injected with the control plasmid pAdTrack(-CMV) that expresses GFP protein only (GFP-1, -2, and -3) and hepatocytes from three mice injected with plasmid pAdTrack-TTRB-T7HisNS5A that co-express GFP and NS5A protein (GFP&NS5A-1, -2, and -3) were harvested for Western blot analysis with antibodies as indicated. D, real time PCR analysis for expression of Aspm gene in mouse hepatocytes. One thousand mouse hepatocytes were captured with a fluorescence detector from each of the group of mouse injected with plasmid pAdTrack-TTRB-T7HisNS5A (GFP+NS5A), and the group was injected with control plasmid pAdTrack(-CMV) (GFP) as indicated. RNA isolation, amplification, and real time PCR analysis of the Aspm gene followed the procedures as described under “Experimental Procedures.” The results shown represent averages from three independent experiments.