FIG. 1.

In vitro inhibition of HBV gene expression by H1- or U6-driven shRNAs. (a) Schematic representation of the shRNA-encoding pAAVEMBL plasmids containing the H1 or U6 promoter. The predicted structures and sequences of the three HBV-specific shRNAs and the control GL2 shRNA are depicted, with the sense strand shown in bold. (b) Inhibition of HBsAg expression by shRNAs. Huh-7 cells were transfected with pHBV1.3 alone or together with the indicated amounts of shRNA-expressing pAAVEMBL-H1 or pAAVEMBL-U6 plasmids for 72 hr, and then HBsAg levels in the culture supernatant were analyzed by enzyme-linked immunosorbent assay. The data are presented as a percentage of that produced by cells transfected with pHBV1.3 alone (mock transfection, mean±SD by three wells per condition). (c) Small RNAs from cells transfected with 1 μg of the different pAAVEMBL-H1 or pAAVEMBL-U6 plasmids transfected were detected by Northern blot using radiolabeled probes identical in sequence to the antisense strand of the corresponding HBV or luciferase shRNAs. 5.8S rRNA was stained with ethidium bromide as a loading control. HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; ITR, inverted terminal repeat; SD, standard deviation; shRNA, short hairpin RNA.