Lentivirus production

Plate ~3.6x106 Lenti-X HEK293T cells (Takara) in 10 cm dish in 10 mL standard DMEM. Cells should be ~80% confluent at the time of transfection.

Next day, remove 5 mL medium and replenish with fresh medium.

Warm up reduced serum medium e.g. Opti-MEM (Gibco) and transfection reagent to room temperature (RT). This protocol was performed with Lipofectamine 3000 transfection reagent (Thermo).

Add 24 µL Lipofectamine 3000 to 600 µL Opti-MEM, mix by vortexing and incubate 5 min at RT.

In another tube, mix 6 µg plasmid containing gene of interest, 5 µg packaging plasmid psPAX2

(RRID:Addgene_12260), 1 µg envelope plasmid pMD2.G (RRID:Addgene_12259) and 24 µL P3000 reagent (provided by manufacturer along with Lipofectamine 3000 reagent) in 600 µL Opti-MEM, mix by vortexing and incubate 5 min at RT.

Mix contents of both tubes and incubate for 15 min at RT.

Add DNA-lipid complex to cells dropwise.

2 days later, collect virus-containing medium and centrifuge for 5 min at 1,000 x g.

Collect supernatant in a fresh tube and proceed with concentration.

Add Lenti-X concentrator (Takara) to clarified virus-containing medium at 1:4 dilution and mix well by gently inverting tube.

Incubate overnight or 2 h at 4 °C.

Next day, centrifuge for 45 min at 1,500 x g at 4 °C followed by gently aspirating supernatant.

Resuspend viral pellet in 100 - 1000 µL PBS, aliquot and store at -80 °C until use.