TITLE: Small scale Lentivirus Production and Infection
AUTHORS: Herschel  Dhekne, Suzanne R R R Pfeffer

[DESCRIPTION]
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. 

[STEPS]
SECTION: Make lentivirus
1. Plate 293T cells at 40% confluency in a 6 well tissue plate submerged under 2 mL medium per well.
SECTION: Make lentivirus
2. After 360 min, most cells will have attached.
SECTION: Day 0
3. 
Prepare DNA mix for transfection:
SECTION: Day 0
3.1. Add the following to 100 µL Optimem per well for transfection:
 
     1µg      PsPAX2 1µg helper plasmid (Addgene ##12260)        0.5µg      VSV-G / pMD2.g (Addgene #12259)         1µg      Lentivirus vector (see below)   

SECTION: Day 0
3.2. Add PEI (from a 1 mg/mL stock) to this mixture solution at ratio 5:1 w/w (PEI:DNA).
Example, 12.5 µg PEI for 2.5 µg DNA mix.

SECTION: Day 0
3.3. Mix DNA mix gently and incubate for 20 min at Room temperature.
SECTION: Day 0
3.4. Add the mix to the cells dropwise.
SECTION: Day 1 (16 hours later)
4. 
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.

 

SECTION: Day 2
5. 
2880 min 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”.
SECTION: Day 2
6. Carefully add an additional 2 mL complete DMEM medium into each well without splashing or disturbing the monolayer.
SECTION: Day 2
7. Bleach all tips and pipettes used to collect the virus.
SECTION: Day 3
8. 
4320 min 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.
SECTION: Day 3
9. The pooled virus (~4 mL) is transferred into a 15ml tube and centrifuged at 250 x g for 5 min.
 

SECTION: Day 3
10. Prepare 0.5 mL aliquots of the lentivirus and freeze at -80 °C.
SECTION: Lentivirus Infection
11.  
Thaw a 0.5 mL virus aliquot in a 37 °C water bath, flicking tube gently to facilitate gentle thaw.
SECTION: Lentivirus Infection
12. Add 1 µL, 10 mg/mL Polybrene.

 

SECTION: Lentivirus Infection
13. 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 4 μg/ml.
SECTION: Lentivirus Infection
14. 2880 min post infection, cells are ready for analysis or selection.
SECTION: Concentrating the virus
15. 
 

SECTION: 4×Lentivirus Concentrator Solution
16. Dissolve 80 g PEG-8000 and 14.0 g NaCl in 80 mL MilliQ water.
SECTION: Virus concentration protocol
21.  
Carefully transfer the virus supernatant into a new 50 ml tube.
SECTION: Virus concentration protocol
22. Add 1 volume of concentrator solution to 3 volumes of virus supernatant (eg. 1 mL concentrator solution for 3 mL virus).
SECTION: Virus concentration protocol
23. Mix by gentle shaking for ~20 s then incubate with constant rocking at least 240 min at 4 °C.
 

SECTION: Virus concentration protocol
24. Spin down at 1600 x g for 60 min at 4 °C.
SECTION: Virus concentration protocol
25. Carefully remove supernatant without disturbing the pellet.
 

SECTION: Virus concentration protocol
26. 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.
SECTION: Virus concentration protocol
27. Aliquot and store at -80 °C until use.

SECTION: Alternative Centrifugation- based Virus concentration method
28.  
 
4320 min after transfection, collect the virus-containing supernatant in a BSL-2+ facility (take only Day 3 supernatant).
SECTION: Alternative Centrifugation- based Virus concentration method
29. Spin down at 250 x g for 5 min at Room temperature.
SECTION: 4×Lentivirus Concentrator Solution
17. Add 20 mL, 10X PBS (pH 7.4).
SECTION: 4×Lentivirus Concentrator Solution
20. Sterilize by passage through a   filter. 
 

SECTION: 4×Lentivirus Concentrator Solution
19. Adjust the final volume to 200 mL.
SECTION: 4×Lentivirus Concentrator Solution
18. Mix with gentle stirring, heating gently only if necessary, until the solids are dissolved then adjust pH to 7.0~7.2.
SECTION: Alternative Centrifugation- based Virus concentration method
30. Transfer the precleared supernatant to ultracentrifuge tubes and pellet at 90000 x g for 90 min at 4 °C.
SECTION: Alternative Centrifugation- based Virus concentration method
32. Make aliquots of 0.2 mL concentrated virus and freeze at -80 °C. 
SECTION: Alternative Centrifugation- based Virus concentration method
31. Remove the supernatant and leave a little less than 1 mL in the tube. Use a 1 mL pipette to recover the remaining pellet which may be difficult to see.