Lentivirus Generation

293FT cells in 75 cm2 flasks – seeded at 6 x 106 cells with 15ml of growth medium
Growth medium: DMEM, 10% FBS, antibiotic – warm to 37°C
Trypsin – warm to 37°C
1X PBS (without calcium) – warm to 37°C
OptiMEM w/ 4% FBS – warm to 37°C
OptiMEM – warm to 37°C
1µg/µl of Packaging Constructs (pRE, pRSV, pMDG)
1µg/µl of Viral DNA (shRNA – GFP Ctrl and target gene)
Lipofectamine 2000 (Invitrogen, Cat# 11668-019)
15 ml tubes, 10% bleach,
PEG-it Virus Precipitation Solution 5X (Systems Biosciences, Cat# LV810A-1

1. Cell Plating
   Seed 293FT  (or LentiX) cells in 75 cm2 flasks at 6 x 106 cells with 15ml of growth medium
   Incubate to confluency (~1-2 days)

2. Transfection
   Slowly aspirate medium and add 5 ml of OptiMEM w/ 4% FBS [NOTE: 293FT cells are very fragile. Set pipettor to the slowest setting and dispense medium by running liquid on the side of the flask without touching the cells.]
   Incubate cells at 37°C until ready for transfection.
   Prepare lipofectamine mix in 15ml tubes. Mix by inversion and incubate for 5 min:
   Lipofectamine 2000: 36µl x (n+0.2)
   OptiMEM: 1.5ml x (n+0.2)
   [NOTE: n = number of viral DNA to be transfected]
   Prepare packaging construct mix in 15ml tubes. Mix by inversion:
   1µg/µl Packaging Construct 1: 3µl x (n+0.2) = 3µg final con’c
   1µg/µl Packaging Construct 2: 3µl x (n+0.2) = 3µg final con’c
   1µg/µl Packaging Construct 3: 3µl x (n+0.2) = 3µg final con’c
   OptiMEM: 1.5 ml x (n+0.2)
   [NOTE: n = number of viral DNA to be transfected]
   Prepare transfection mix in 15ml tubes. Mix by inversion and incubate for 20 min :
   

packaging construct mix: 1.5 ml 
1µg/µl viral DNA (shRNA): 3µl = 3µg final con’c
lipofectamine mix: 1.5 ml
Slowly dispense (as described above) 3 ml of transfection mix to the 293FT cells.
Incubate overnight

3. Transfection (con’t)
   Slowly (as described above) aspirate medium and dispense 20 ml of growth medium to the 293FT cells [NOTE: Wash with bleach all disposables that came in contact with the transfected media]
   Ensure that all cells are still attached after changing medium then Incubate for 48h

4. Collection of Virus
   Check cells under microscope [cells successfully expressing viruses should have large bubble-like structures attached to them, some cells may die during the process]
   Slowly (as described above) collect supernatant unto 50 ml tubes [NOTE: Wash with bleach all disposables that came in contact with the transfected media]. – Virus may be stored in 4°C prior to processing 
   Centrifuge at max speed (5000 rpm) 
   Decant supernatant unto 30 ml syringe and filter through 0.45µm (low protein binding) filter unto new 50 ml tubes 
   Add 4.8ml of PEG-it Virus Precipitation Solution (1 volume per 4 volume of viral supernatant) 
   Mix by inversion and Incubate at 4°C overnight (at least 12 h in refrigerator) [stable upto 2 days] 
   Centrifuge supernatant / PEG-it mixture at 1500 x g for 30 min at 4°C 
   Aspirate supernatant. Spin down residual PEG-it solution by centrifugation [viral pellet = beige/white pellet] 
   Resuspend pellet in 1/10 to 1/100 of original volume using cold, sterile PBS or DMEM containing 25 mM HEPES buffer at 4°C 
   Aliquot and store at -70°C