For transfections, a full-length linear HBV DNA (nt 1820-1820) with sticky ends was used.
As previously described, linear HBV monomers were excised from the plasmids by restriction enzyme digestion with 5 U of BspQI (New England Biolabs, Beverly, MA, USA) at 50 °C. The 3.2 kb fragments were gel purified with PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions and the DNA was quantified spectrophotometrically [20 (link)].
For transfections, cells were seeded in 6 or 24 well plates and grown to 60–70% confluence. Transfections were carried out using X-tremeGene 9 transfection reagent (Roche, Mannheim, Germany), according to the manufacturer’s recommendations. Transfection with a linear full-length HBV genome derivative from the pCH-9/3091 POL minus plasmid was used as control for evaluating the amount of remaining input DNA. For trans-complementation assays, cells were co-transfected with sgtF1b X(-) or sgtF4 X(-) genomes and sgtF1b HBx or sgtF4 HBx expression plasmids, respectively. Cells were maintained at 37 °C in 5% CO2 atmosphere. After 6 h incubation, medium was replaced, and cultures were incubated for 72 or 96 h. In order to eliminate HBV DNA input, cell culture medium was collected every 24 h, cells were washed six times with PBS, and fresh medium was added.
As previously described, linear HBV monomers were excised from the plasmids by restriction enzyme digestion with 5 U of BspQI (New England Biolabs, Beverly, MA, USA) at 50 °C. The 3.2 kb fragments were gel purified with PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions and the DNA was quantified spectrophotometrically [20 (link)].
For transfections, cells were seeded in 6 or 24 well plates and grown to 60–70% confluence. Transfections were carried out using X-tremeGene 9 transfection reagent (Roche, Mannheim, Germany), according to the manufacturer’s recommendations. Transfection with a linear full-length HBV genome derivative from the pCH-9/3091 POL minus plasmid was used as control for evaluating the amount of remaining input DNA. For trans-complementation assays, cells were co-transfected with sgtF1b X(-) or sgtF4 X(-) genomes and sgtF1b HBx or sgtF4 HBx expression plasmids, respectively. Cells were maintained at 37 °C in 5% CO2 atmosphere. After 6 h incubation, medium was replaced, and cultures were incubated for 72 or 96 h. In order to eliminate HBV DNA input, cell culture medium was collected every 24 h, cells were washed six times with PBS, and fresh medium was added.
Free full text: Click here
