One important issue is confirming that the gene or protein-of-interest is expressed in these cells prior to the knock-down experiments. In preliminary studies we have determined that there is baseline expression of both ISG15 and UBP43 mRNA in both Huh7.5 and replicon cells (real-time PCR) and that IFNa strongly induces protein expression of ISG15 in Huh7.5 cells. These results argue that the ISG15 and UBP43 pathway is relevant to this model.