Lentiviral and Retroviral Vector Production

As described previously (30 (link)), 1 day before transfection, 293T packaging cells were plated into 6-well plates at a density of 6 × 10⁵ cells/well. Cells were cotransfected 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) according to the manufacturer's instructions. One day after transfection, cells were refed with fresh medium containing 30% (v/v) FBS. After 24 hours, medium containing virus was harvested, passed through 0.45 μmol/L 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).