Production of lentiviruses in Lenti-X cells
F Ulrich Hartl, Cole S Sitron
Abstract
This is a protocol to produce lentivirus in a HEK cell line selected to produce high-titer virus.
Attachments
Steps
Transfection
Plate Lenti-X cells 2.5 x 10^6/10 cm 24h 0m 0s before transfection.
Warm OptiMEM, X-tremeGENE HP DNA Transfection Reagent, and DNA to warm to Room temperature.
Add 1mL OptiMEM to a tube.
Add plasmid DNA to the OptiMEM, gently pipetting to mix.
| A | B | C |
|---|---|---|
| psPAX2 | 0.65 pmol | 4600 ng |
| pMD2.G | 0.36 pmol | 1400 ng |
| Transfer vector | 0.82 pmol |
Add X-tremeGENE HP DNA Transfection Reagent to OptiMEM/DNA (4µL per 1µg of DNA).
Incubate for 0h 15m 0s at Room temperature.
Add OptiMEM/DNA/X-tremeGENE to the plate of cells dropwise, then gently swirl the plate.
The day after transfection, replace the medium with
Collection
72h 0m 0s after transfection, collect the medium in a 15 ml conical.
Centrifuge 500x g,0h 0m 0s x 0h 5m 0s, then pass the supernatant through a .22 µm filter.
Add 3mL of LentiX concentrator to the supernatant, invert 6 times to mix, then incubate 0h 45m 0s at 4°C.
Centrifuge at 1500x g,0h 0m 0s for 0h 45m 0s at 4°C.
Remove the supernatant and resuspend the pellet in 120µL ice cold PBS.
Make aliquots in multiples of 14µL and freeze at -70°C.
