Large-Scale Lentiviral Vector Production

Pooled library virus was made using the same large scale T175 flask method used previously [19 (link)]. Briefly, 24 hours pre-transfection, 18 × 10⁶ HEK293T cells were seeded into a 175 cm² tissue culture flask with 24 mL of DMEM + 10% FBS. Next day, one solution of Opti-MEM (Corning, 6 mL) and LT1 (Mirus, 305 μL) was combined with a DNA mixture of the packaging plasmid pCMV-VSVG (Addgene 8454, 5 μg), psPAX2 (Addgene 12260, 50 μg), and sgRNA-containing vector (pPapi, 40 μg). This mixture was incubated for 20–30 min at room temperature, during which media was changed on the HEK293Ts. Following incubation, the transfection mixture was added dropwise to cells. The cells were incubated for 6–8 h, after which time media was replaced with DMEM + 10% FBS, supplemented with 1% BSA. 36 hours post-media replacement, virus was harvested.