Lentiviral Vector Production and Transduction

Lentivirus was produced by transient transfection of 293T (ATCC) cells using linear 25-kDa polyethyleneimine (PEI; Polysciences). Briefly, 4 × 10⁶ cells were plated onto 10-cm tissue culture plates. After 24 h, 3 µg of psPAX2, 1.5 µg of pMD2G (Addgene plasmid #12260 and #12259, respectively) and 6 µg of lentiviral vector plasmid were mixed in 500 µl diluent (5 mM HEPES, 150 mM NaCl, pH = 7.05) and 42 µl of PEI (1 mg/ml) and incubated for 15 min. The DNA/PEI complex was then added to the plate drop-wise. Lentivirus was harvested 48 h post-transfection, concentrated 100-fold by low-speed centrifugation at 8000g for 18 h and titered on human Nalm6 cells by flow cytometry and qPCR as previously described (20 (link)). Transduction of the target cell line was carried out in six-well plates containing 2 × 10⁶ cells per well in 2 ml of growth media and 4 µg/ml hexadimethrine bromide (Polybrene; SIGMA). Titered virus was added to each well at the designated multiplicity of infection (MOI; four wells per condition) and the cells were incubated with shaking (130 rpm) at 37°C, in 8% CO₂ for 4–6 h. The cells were then harvested, pooling the replicate wells, and pelleted at low speed (1000g). Transduction media was removed and replaced with 20 ml fresh media and cells were transferred to a 125-ml vented shake flask (Nalgene). Copy number was estimated using a previously established genomic Q-PCR-based assay with comparison to a housekeeping gene as well as control cell lines with a defined viral integration number based on Southern blotting (21 ).