Small scale Lentivirus Production and Infection
Suzanne R Pfeffer, Herschel Dhekne
Abstract
This protocol can be used for production and transduction of lentiviral sgRNA, shRNA and protein overexpression in conjunction with generation 2 and generation 3 lentivirus plasmids.
Attachments
Steps
Make lentivirus
Plate 293T cells at 40% confluency in a 6 well tissue plate submerged under 2mL medium per well.
After 6h 0m 0s, most cells will have attached.
Day 0
Prepare DNA mix for transfection:
Add the following to 100µL Optimem per well for transfection:
| A | B |
|---|---|
| 1 µg | PsPAX2 1µg helper plasmid (Addgene ##12260) |
| 0.5 µg | VSV-G / pMD2.g (Addgene #12259) |
| 1 µg | Lentivirus vector (see below) |
Add PEI (from a 1mg/mL stock) to this mixture solution at ratio 5:1 w/w (PEI:DNA).
Example, 12.5µg PEI for 2.5µg DNA mix.
Mix DNA mix gently and incubate for 0h 20m 0s at Room temperature.
Add the mix to the cells dropwise.
Day 1 (16 hours later)
Check for cell viability; at this time, >70% of the cells should be transfected and virus is already being produced and is being released into the supernatant.
Day 2
48h 0m 0s after transfection, collect the culture supernatant in a BSL-2+ facility; centrifuge in an enclosed rotor and remove supernatant with care. This is “Day-2 virus”.
Carefully add an additional 2mL complete DMEM medium into each well without splashing or disturbing the monolayer.
Bleach all tips and pipettes used to collect the virus.
Day 3
72h 0m 0s after transfection, collect the culture supernatant in BSL-2+ facility as before. This is “Day-3 virus". Day-2 and Day-3 virus are then pooled; Day-2 titre is lower than Day-3.
The pooled virus (~4mL) is transferred into a 15ml tube and centrifuged at 250x g,0h 0m 0s for 0h 5m 0s.
Prepare 0.5mL aliquots of the lentivirus and freeze at -80°C.
Lentivirus Infection
Thaw a 0.5mL virus aliquot in a 37°C water bath, flicking tube gently to facilitate gentle thaw.
Add 1µL, 10mg/mL Polybrene.
Transfer virus mixture to the medium covering 1 well of a 6 well plate containing the target cell line. Polybrene will become diluted in the cell medium to a final concentration of 4μg/ml.
48h 0m 0s post infection, cells are ready for analysis or selection.
Concentrating the virus
4×Lentivirus Concentrator Solution
Dissolve 80g PEG-8000 and 14.0g NaCl in 80mL MilliQ water.
Add 20mL, 10X PBS (7.4).
Mix with gentle stirring, heating gently only if necessary, until the solids are dissolved then adjust pH to 7.0~7.2.
Adjust the final volume to 200mL.
Sterilize by passage through a 0.2 µm filter.
Virus concentration protocol
Carefully transfer the virus supernatant into a new 50 ml tube.
Add 1 volume of concentrator solution to 3 volumes of virus supernatant (eg. 1mL concentrator solution for 3mL virus).
Mix by gentle shaking for ~0h 0m 20s then incubate with constant rocking at least 4h 0m 0s at 4°C.
Spin down at 1600x g,0h 0m 0s for 1h 0m 0s at 4°C.
Carefully remove supernatant without disturbing the pellet.
Thoroughly resuspend the viral pellet in PBS or desired medium using 1/10~1/20 of the original volume by gentle pipetting using a 1ml Pipetman.
Aliquot and store at -80°C until use.
Alternative Centrifugation- based Virus concentration method
72h 0m 0s after transfection, collect the virus-containing supernatant in a BSL-2+ facility (take only Day 3 supernatant). Spin down at 250x g,0h 0m 0s for 0h 5m 0s at Room temperature.
Transfer the precleared supernatant to ultracentrifuge tubes and pellet at 90000x g,0h 0m 0s for 1h 30m 0s at 4°C.
Remove the supernatant and leave a little less than 1mL in the tube. Use a 1 mL pipette to recover the remaining pellet which may be difficult to see.
Make aliquots of 0.2mL concentrated virus and freeze at -80°C.
