The protocol for lentivirus production and transduction have been described elsewhere35 (link), 36 (link). In brief, lentivirus was produced by triple transfection of HEK 293 T cells. Packaging 293 T cells were plated in 10-cm plates at a cell density of 5 × 106 a day prior to transfection in DMEM containing 10% heat-inactivated fetal bovine serum. 293 T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer’s protocol. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 108 to 1 × 109 infectious units per ml. Viral supernatant was collected for three days by ultracentrifugation and concentrated 100-fold. Titers were determined on 293 T cells. Cells were transduced with lentiviral particles expressing gene of interest.
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