Lentiviral and Retroviral Transduction of Human Cells

As previously described (30 (link)), one day before transfection, 293T packaging cells were plated into 6-well plates at a density of 6 × 10⁵ cells/well. Cells were co-transfected with 1 μg of expression plasmid, 1 μg of psPAX2, and 200 ng of VSV-G for lentivirus packaging, or with 1 μg of pMSCV, 1 μg of pMD-MLV, and 200 ng of VSV-G for retrovirus packaging, using TransIT-LT1 Transfection reagent (Mirus Bio, Madison. WI, USA) according to the manufacturer’s instructions. One day after transfection, cells were re-fed with fresh medium containing 30% (v/v) FBS. After 24 hours, medium containing virus was harvested, passed through 0.45 μm cellulose acetate membrane filters, and used fresh for infection. MM cells were spinocculated with viral supernatant in the presence of 8 μg/ml polybrene at 800× g for 30 minutes at room temperature and further infected in 5% CO₂ at 37°C for 5 hours. After 24 hours, lentivirus-infected cells were selected with puromycin dihydrochloride (Sigma-Aldrich) at 1 μg/ml for 2 days. For the generation of OPM2-TurboGFP-Luc, TurboGFP-Luc expressing cells were selected using a cell sorter (BD FACSAria III; BD Biosciences). The human MYC expression vector was constructed by amplifying MYC cDNA using PCR and inserting into the pLenti-DDK-P2A-Puro empty vector (OriGene Technologies, PS100092).