2.5. Generation of HCV Replicon Cell Lines

The FLuc-JFH1/RLuc stable replicon cell line was generated as described previously [44]. To this end, a single-cell suspension of 6 × 10⁴ Huh7-Lunet/RLuc cells was prepared by trypsinization and washed once with phosphate-buffered saline (PBS). Cells were resuspended in 400 μL of Cytomix [45] containing 2 mM ATP and 5 mM glutathione and mixed with 1 μg of in vitro transcribed pFKI389Luc-ubi-neo/NS3-3′_dg_JFH RNA before being transfected by electroporation with a Gene Pulser system (Bio-Rad, Hercules, CA, USA) in a cuvette with a gap width of 0.4 cm (Bio-Rad, Hercules, CA, USA) at 975 μF and 270 V, as described previously [41]. Cells were plated in a T75 Flask and, three days post-electroporation, G418 was added to a final concentration of 750 μg/mL in order to allow selection of a mixed cell population stably replicating HCV genome.