15 cm tissue culture dish

Human embryonic kidney cells (HEK293T) were seeded at a density of 1.5 × 10⁷ cells/15-cm tissue culture dish (Corning) after coating the dishes with 0.01% poly-l-lysine (Sigma). The HIV-1-based lentiviral vector stocks were produced by co-transfecting the helper constructs pLP1, pLP2, and pLP/VSVG [26 (link)] and the transducing plasmid pHAGE NFkB-TA-LUC-UBC-GFP-W (a gift from Darrell Kotton (Addgene Plasmid #49343)) [27 (link)]. SIM-A9 cells were transduced with the harvested vector pHAGE NFkB-TA-LUC-UBC-GFP-W in the presence of 8 μg/ml protamine sulfate (MP Biomedicals, Santa Ana, CA) and incubated at 37° C overnight [28 (link)]. Cells were washed, spun for 5 min at 1000 rpm, and used for FACS analysis followed by sorting the GFP-positive vector transduced cells (Canto-II, BD InFlux cell sorter; BD Biosciences, San Jose, CA). Sorted cells were washed twice prior to expansion.

Human embryonic kidney 293T cells were seeded at a density of 1.5 × 10⁷ cells per 15-cm tissue culture dish (Corning, NY, USA) and precoated with 0.01% poly-l-lysine (Sigma, MO, USA). The integrating viral vector stocks were produced by using the helper constructs pLP1, pLP2 and pLP/VSVG (31 (link)). The non-integrating viral vector stocks were produced by using the helper constructs pCD/NL2 ΔInt, pLP2 and pLP/VSVG plasmids. The lentiviral transfer vector plasmids pLVCG and pLV-S/MAR were co-transfected with the helper constructs to produce integrating and non-integrating vector stocks. As previously described (30 (link)), calcium phosphate transfection was performed in the presence of DMEM (Gibco), 10% FBS (Gibco) and 1% penicillin/streptomycin (Pen/Strep, Gibco). Vector supernatant was harvested 36, 48 and 72 h later, filtered through a 0.45-µm filter, pooled and ultra-concentrated over 30 h at 7300 RCF, and the pellet was resuspended in Iscove’s media (Gibco) and stored at −86°C until use. Limiting dilution titers were determined by FACS and calculated using 293T cells, as previously described (31 (link)). For transduction, cells were washed and resuspended in corresponding media (described below), with 8 µg/ml protamine sulfate (MP Biomedicals). Transductions took place overnight at 37°C followed by wash and culture in DMEM.