Protocol for Retrovirus Production & Transduction

Dominik Haudenschild, May 13, 2009 From Jasper Yik

Materials

Vectors: pMSCV puro (inserts cloned into Xho I and EcoR I), pVSVG

Packaging cell line: GP2-293 (grown in DMEM/hi glucose, 10% FBS, Pen/Strep)

Reagents: Lipofectamine, PLUS reagent, Polybrene.

Packaging virus

1. Before transfection, plate GP2-293 cells on 6-well plate (or scale up as needed) at ~80% confluency. (80% is important as cell death is seen if plating is too dilute (toxicity from transfection), or too crowded, (cell peel off))

2. In general, start transfection in the morning, then replace transfection medium with fresh complete medium 6 hours post-transfection.

3. Transfect GP2-293 cells with the following steps. (for 1 well of 6-well plate)

a) Mix a total of 1.3 ug DNA (use viral vector and pVSVG plasmid in a 4:1 ratio) and 2.25ul PLUS reagent in 77 ul DMEM (no FBS), (add DNA to DMEM first, vortex, then add PLUS) incubate at RT for 15 min.

b) Mix 1.8 ul of Lipofectamine in 77 ul DMEM (no FBS), incubate at RT for 15 min.

c) Combine solutions in a) and b), mix gently and incubate at RT for 15 min.

d) Remove the growth medium from cells and replace with 1 ml of DMEM (10% FBS) without P/S for each well (15ml of media if using 15cm plate).

e) Add the complex from step C to the well, mix gently and incubate at 37℃ in CO₂ incubator for ~6-8 hours. Then remove the medium and add 1 ml of fresh DMEM (10% FBS, 1X P/S).

4. 48 hours after transfection, harvest the viral supernatant into a 1.5 ml tube. Spin at 500 g for 5 min, and filter the supernatant with 0.45 um filter (low protein binding).

5. Freeze virus in liquid nitrogen and store at -80℃. (Avoid multiple freeze-thaw cycles)

Concentrating virus

1. Sterilize the 5 ml ultra centrifugation tubes (Beckman 344057) with 70% ethanol 20 min.

2. Dry the tubes on a clean wipe in hood.

3. Pipet 5 ml viral supernatant to centrifuge tube, place the tube into centrifuge bucket gently. Make sure the lid of bucket was sterilized with 70% ethanol.

4. Put buckets onto sw55Ti rotor, make sure they are in right position.

5. Spin at 23k rpm, 90 min, 25℃.

6. Aspirate as much supernatant as possible, add desired volume of complete medium and gently resuspend virus. Transfer virus to a sterilized 1.5ml tube. Incubate tube at 4℃ overnight.

Infecting target cells

1. Before infection, plate your target cells on 6-well plate at ~70-80% confluency.

2. Add polybrene to the concentrated viral supernatant to a final concentration of 5 ug/ml.

3. Remove the medium from cells and replace with 1ml of viral supernatant which contains 5 ug/ml polybrene for each well, incubate at 37℃ in CO₂ incubator overnight.

4. Remove the viral supernatant and replace with 2 ml of fresh medium to each well, incubate at 37℃ in CO₂ incubator.

5. 48 hours after infection, harvest cells to detect the infection efficiency or continued to do other experiments.