Production and usage of VSV-G pseudotyped lentiviruses

Lentiviral Packaging, Concentration, and Transduction 

Introduction: This protocol describes the production, concentration, and usage of second generation VSV-G pseudo typed lentiviruses for gene delivery in mammalian tissue culture.

Growing Packaging Cells: Grow 293T viral packaging cells in DMEM containing 4.5 g/L glucose and 110 mg/mL sodium pyruvate, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. Passage 90% confluent cultures by trypsinization and re-seed 1:5 or 1:10 dilutions for re-splitting every 2-3 days. As a rule of thumb use a multiplication factor of 2.5 per day when cells are 90% confluent.

Materials

• 293T culture
• DMEM (Gibco, 11995073)
  • + 10% FCS (Genclone, 25-550)
  • + 1% P/S (Gibco, 15140122)
• Trypsin-EDTA 0.25% (Gibco, 25200072)
• 1M HEPES (Gibco, 15630080)
• Polybrene (Sigma, TR-1003)
• Lipofectamine 2000 (Invitrogen, 11668019)
• OptiMEM-I (Gibco, 31985070)
• Amicon Ultra Centrifugal Filter, 100 kDa MWCO (Milipore UFC910008)
• 0.45 µm PVDF syringe filters (Genclone, 25-242)
• 10 mL Luer-Lock syringes (BD, 302995)
• T75 flasks
• 6-well plates
• 15 mL conical tubes
• 2nd generation viral packaging plasmids (vsv-g, gag-pol-tat-rev)

• Desired lentiviral transfer vector

   

Day 1: Seed cells for transfection

1. 18-24h before transfection, trypsinize and gently resuspend a culture of 293Ts in fresh DMEM
2. Seed 6E6 cells in a T75 flask (one per vector to be prepped) in 10 mL of DMEM
3. Swirl flasks in a 12-6 and 9-3 direction several times to allow cells to attach evenly on flask surface
4. Culture the flask horizontally overnight in a 37°C / 5% CO₂ humidified incubator

Day 2: Transfect cells with viral packaging plasmids

1. Cells should be ~70% confluent today: check that they are not too confluent before proceeding
2. Prepare transfection mixes as follows (for each T75)
3. 2 µg VSV-G vector (encoding vsv-g)
4. 5 µg ΔR8.91 vector (encoding gag-pol-tat-rev)
5. 2.5 µg of your desired lentiviral vector
6. In a 15mL conical tube (per transfection), combine 1.5 mL of room temp OptiMEM-I (Invitrogen) and 90µL of Lipofectamine 2000 (Invitrogen). Flick gently to mix and incubate 5 min at RT
7. In a separate 15 mL conical tube combine 1.5 mL of room temp OptiMEM-I (Invitrogen) with packaging mix from Step 2 above
8. Combine the mixture from Step 3 and Step 4, mix quickly but gently by flicking, and incubate 20 min at RT
9. Aspirate growth media from the flask of 293Ts, and replace with 7mL of warm DMEM
10. After forming transfection complexes for 20 min, add the resulting 3mL of transfection mix gently dropwise around the flask. The cells are loosely adherent: be gentle
11. Return flasks to the incubator until the next day 

Day 3: Add viral collection medium and seed target cells

1. 24 h after the transfection, replace the transfection medium with 10 mL of virus collection medium (DMEM growth medium supplemented with 15 mM HEPES)
2. Return flasks to the incubator and grow another 24 h
3. If you are planning to use freshly collected virus to infect your target cells, seed your target cells into 6-well plates (one per infection) to be ready to transduce in 24 h. Aim for 60% confluency on the day of transduction

Day 4: Collect lentiviral supernatant, concentrate and store, or infect target cells

1. 24 h after adding viral collection medium (48 h after transfection) collect viral supernatant. If two collections are desired, store the first collection at 4°C, add more medium to the packaging cells and collect a 2nd time 24 h after the first collection (72 h after transfection). Pool the harvests together.
2. Filter the viral supernatants slowly through a 0.45 µm PVDF low protein-binding syringe filter unit to remove 293T cell debris prior to usage or concentration and storage
   
   NOTE: Recombinant lentiviruses are extremely sensitive to pH changes, and it is critical that the media not be yellowed at the collection stage. If this occurs, increase the volume of viral collection medium, or titrate down the amount of FCS or glucose in the growth media
3. Apply the filtered viral supernatants to a regenerated cellulose centrifugal filter unit with a 100K MW cut-off (Amicon). DO NOT overflow the chamber (15 mL max)
4. Centrifuge in swinging bucket centrifuge in the tissue-culture room at 1500 xg in aerosol-resistant canisters outfitted with 50 ml conical tube holders. Check filtrates every 5 minutes until you reach a concentration of 10-20 X
5. Discard and bleach the eluate and use the filtrate (concentrated virus) to immediately infect target cells or store at 4°C for up to a week. You can also snap freeze aliquots in liquid nitrogen and store at -80°C. A drop in titer is expected when freezing
6. To infect target cells, add growth medium and viral supernatant totaling 2 mL to each well. Supplement with polybrene to a final concentration of 8 µg/mL
   1. If using concentrated virus, you may choose to determine titer. If you choose not to titer, aim to infect x2 wells of a 6-well for the virus generated from every T75 flask
7. Seal 6-well plates with parafilm around edges, then centrifuge for 2 h at 700 xg in an aerosol resistant microplate carrier. If possible, centrifuge at 37°C
8. Return plates to incubator, and grow overnight 

Day 5: Expansion of transduced cultures

1. The day after transduction, wash cells x2 times with PBS, then trypsinize and transfer all cells from x1 well of a 6-well to x1 10cm dish. Grow for another 48 h before beginning antibiotic selection or expanding for FACS based enrichment of transduced populations

Notes:

1. VSV-G vector was obtained from Clontech
2. ΔR8.91 vector was a gift from Brian Rabinovitch, MDACC