Cell culture and transfections. Huh7
Dorsomorphin were maintained in DMEM (Sigma–Aldrich, MI, USA) supplemented with 2.5% fetal calf serum (FCS), 50 IU/ml penicillin and 50 mg/l streptomycin under standard conditions. Cells were plated in 24-well plates (Nunclon?, Nunc, Denmark) at a cell density of 1 × 106 cells per well. Transfections were carried out using Lipofectamine?-2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions and were performed when cells reached approximately 70% confluency. To assess stability of linear cassettes and duration of transgene expression, LIN-FLuc, ITR-FLuc and pCMV FLuc cassettes were transfected into Huh7 cells. On the third day after transfection, the cells were harvested by trypsinization. Half were re-plated and the remainder was processed for assay of Firefly luciferase activity using the Dual-Luciferase Reporter? Assay System (Promega, WI, USA). Assays of Firefly luciferase activity were carried out similarly at days 5, 7, 9, 11 and 13 after transfection. eGFP expression was detected by employing fluorescence microscopy, which was carried out 48 h after using similar transfection protocols. To determine the HBsAg knockdown efficacy of the linear and ITR cassettes, Huh7
cells were cotransfected with 100 ng of pCH-3/3091 and 1 μg of either LIN-122/5 or ITR-122/5. The concentrations of HBsAg in culture supernatants were measured 48 h after transfection by ELISA using the MONOLISA? HBs Ag ULTRA kit (Bio-Rad, CA, USA).
