TITLE: Generation of stable cell lines using viral infection
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for generation and precipitation of retrovirus, and infection of HeLa cells to generate stable cell lines.

[STEPS]
SECTION: Day 1
1. Seed HEK293T cells into a 10cm tissue culture plate (6.1 million cells/plate), seeding one plate per construct you are generating virus for. 
SECTION: Day 2
2. Transfect cells with viral and helper vectors using lipofectamine LTX. In a 15mL falcon tube, combine the following:
  
            Reagent      Amount        1      Viral vector construct containing cDNA of interest      9 mg        2      Gag-pol vector      6 mg        3      VSV-G vector      3 mg        4      Opti-MEM      3 mL   


SECTION: Day 2
3. Add 18 µLand vortex for ~20 s. Incubate at Room temperature for 5 min. 

SECTION: Day 2
4. Add 54 µL, vortex for ~20 s. Incubate at Room temperature for 20 min. 

SECTION: Day 2
5. Once the 20 minute incubation starts, replace the media on the HEK cell plates with 7 mL.

SECTION: Day 2
6. At the end of the incubation, add the lipofectamine/OptiMEM mix to the plate using a sterile transfer pipette, in a drop wise fashion across the plate. Gently rock the plate to mix, and return the plate to the incubator. 

SECTION: Day 3
7. In the morning, remove the media from the HEK cell plate and dispose of the media in a beaker containing bleach. Add 6 mL of fresh growth media to the HEK cell plate. 

SECTION: Day 4
8. In the late afternoon, collect the media from the HEK cell plate into a falcon tube. Add back8 mLfresh growth media to the HEK cell plate. 

SECTION: Day 4
9. Centrifuge the media collected at maximum speed for 5 min at Room temperature to pellet any debris. Pass the media through a 0.45 mm syringe filter into a clean falcon tube. Parafilm the falcon tube, and place store the media at 4 °C in the fridge. 

SECTION: Day 6
10. In the morning, collect the media from the HEK cell plate into a falcon tube. Centrifuge the collected media at maximum speed for 5 min atRoom temperature. Pass the media through a 0.45 mm syringe filter into a clean falcon tube. 

SECTION: Day 6
11. Combine the media collected in step 9 and in step 10 into one tube. Add Lentivirus Precipitation Solution (ALSTEM) to the media at a ratio of 1:4 (Lentivirus precipitation solution : viral media).
SECTION: Day 6
12. Gently invert the tube 8 – 10 times to mix the precipitation solution and media together. Parafilm the tube and leave on a rocker gently rocking at 4 °C for 2880 min.

SECTION: Day 7
13. If intending to infect cells with fresh virus, seed the cells to be infected into a 6 well plate (~220K cells/well).  
SECTION: Day 8
14. After the 48 hour rocking incubation, centrifuge the viral precipitation mix at 1600 rcf, 4 °C for 60 min

SECTION: Day 8
15. Remove the supernatant, disposing of it into a beaker containing bleach. Resuspend the precipitated pellet in 3 – 6 mL of fresh growth media (3 mL if you require high construct expression). 
SECTION: Day 8
16. Replace the media on the cells to be infected with1.5 mL fresh growth media/well. Add 500 µLof the precipitated virus to each well to be infected. 

SECTION: Day 8
17. Add polybrene at a final concentration of 8 mg/mL to each well, and rock the plate to mix. Place the cells back into the incubator. 
SECTION: Day 8
18. Any leftover virus can be frozen at -80 °C for use at a future date 

SECTION: Day 9 - 10
19. After 24 – 48h (depending on the construct and expression required), remove the media from the infected cells and place in bleach to dispose. Add fresh growth media back to the cells. 
SECTION: Day 15 - 17
20. After 5 - 7 days after the virus has been removed (and after three passages), using FACS, sort the cells populations to the desired fluorescent expression level.