Genjet ver 2 transfection reagent

For lentivirus production, 293T cells were co-transfected with the lentiviral transfer vector together with the packaging plasmid psPAX2 and the vesicular stomatitis virus G protein expression plasmid pVSV-G at a ratio of 5:4:1 using GenJet (Ver. II) transfection reagent (SignaGen). Two days later, virus was collected from the medium by ultracentrifugation in an SW28 rotor at 25,000 rpm for 2 h at 4°C. Virus pellets were resuspended in an appropriate volume of PBS to achieve 100x concentration. The transduction unit of lentiviruses was determined in 293T cells in the presence of appropriate antibiotics. To produce infectious BAC16 viruses, iSLK-BAC16 cells were reactivated with 1 μg/ml doxycycline and 1 mM sodium butyrate for 3 days. Infectious titer of BAC16 virus in the culture supernatant was determined by spinoculation of 293A cells and 1 day later GFP-positive cells were quantified using flow cytometry. BJAB cells were infected with 1 infectious unit of BAC16 by spinoculation at 800 x g for 30 min at room temperature. For lentiviral transduction of GFP, MAVS rgRNA1, and K118-only vFLIP (in pDUET110) into BCBL-1 cells, cells were incubated with 1 transduction unit (TU) in the presence of 5 μg/ml polybrene for 1 day and washed in complete RPMI 1640 media, and further cultured for cell viability assays and immunoblotting assays.