Lentiviral Particle Production in HEK293T

Lentiviral particles were produced in HEK293T cells as described in (Azzarelli et al., 2018a (link)). Briefly, HEK293T cells were transfected with third generation packaging vectors (vectors containing VSVG, gag-pol, rev and tat in a ratio 7:1:1.1) and with pBob-H2B-GFP vector (kind gift from Professor Rick Livesey), using Calcium Phosphate method (Promega ProFection^® kit, E1200). The morning after transfection, the medium was refreshed and then collected for viral production 36 h later. To concentrate the viral particles, the medium was mixed with the concentrator (Lenti-X™ Concentrator, Clontech, 63123) overnight at 4°C and then spun for 45 min at 2000× g at 4°C. After removing the supernatant, the pellet containing viral particles was resuspended in 200–300 µl of medium and stored in 20 µl aliquotes at −80°C. Viral titer was quantified using Titration kit (Lenti-X™ Titration kit, Clontech, 631235); the viral particles needed have been calculated using the formula: viral particle=[(total number of cells×MOI-multiplicity of infection)/titer)]×1000. The MOI used in our experiments is 10.