Lentivirus production protocol

A. Transfection in 10cm dish

  1. Day 1. Seed 293FT cell into 10cm dish, ~1e7/well
  2. Day 2. Cells grow to ~90% confluence, perform transfection with Lipofectamine 2000
    1. Mix 1. Dilute 50ul Lipofectamine 2000 in 1.5ml Opti-MEM, mix well, incubate for 5min at RT.
    2. Mix 2. Dilute the following plasmids in 1.5ml Opti-MEM, mix well, and incubate for 5min at RT.
      1. 5.6ug pMD2.G
      2. 14ug Target vector
      3. 8.4ug psPax2
    3. Mix 1 and 2, then incubate for 10min at RT;
    4. Aspirate old medium, add 8ml serum-free medium, and add transfection mix into cells;
    5. After 4-5 hours, change to 8ml fresh complete DMEM medium.
  3. Day 4. Collect the 1st viral supernatant into 15ml tubes and concentrate with lenti-X concentrator, add 8ml fresh DMEM medium to cells, and continue culturing;
  4. Day 5. Collect the 2nd viral supernatant and discard the packaging cell, and concentrate the 2nd viral supernatant with Lenti-X concentrator.

 

B. Transfection in 6-well plate

  1. Day 1. Seed 293FT cell into 6-well plate, ~1e6/well
  2. Day 2. ~90% confluence, transfection with Lipofectamine 2000
    1. Mix 1. Dilute 10ul Lipofectamine 2000 in 125ul Opti-MEM, mix well, incubate for 5min at RT.
    2. Mix 2. Dilute the following plasmids in 125ul Opti-MEM, mix well, and incubate for 5min at RT.
      1. 0.9ug pMD2.G
      2. 2.3ug Target vector
      3. 1.4ug psPax2
    3. Mix 1 and 2, then incubate for 10min at RT;
    4. Aspirate old medium, add 1.5ml serum-free medium, and add transfection mix into cells;
    5. After 4-5 hours, change to 2ml fresh complete DMEM medium.
  3. Day 4. Collect the 1st Viral supernatant into 15ml tubes and concentrate with Lenti-X concentrator, add 2ml fresh medium to cells and continue culturing;
  4. Day 5. Collect the 2nd viral supernatant and discard the packaging cell, and concentrate the viral supernatant as previously described.

 

Virus concentration protocol

  1. Centrifuge viral supernatant at 500g for 10min.
  2. Transfer the supernatant into a new 15ml tube, and discard cell debris.
  3. Add Lenti-X concentrator into viral supernatant at a 1:3 ratio, and mix by gently inverting.
  4. Incubate at 4C for 30min to 24h (minimum 30 min, maximum 1 week).
  5. Centrifuge at 1500g, 4℃ for 45min.
  6. Discard supernatant.
  7. Re-suspend viral pellet with 500ul-1ml PBS.
  8. Aliquot into small volume and store at -80C until use.