Generation of stable cell lines using retroviral system
nguyen.tha
Abstract
This protocol details generation of stable cell lines using retroviral system.
Attachments
Steps
Day 1
Seed NIH HEK293T cells into a 6-well plate (900k cells/well if set up in the morning, 950k cells/well if set up in the afternoon).
Day 2: The following protocol is designed for one well of the 6-well plate
Transfect cells with viral and helper vectors using lipofectamine LTX. Combine the following in a 1.5 mL tube:
| A | B |
|---|---|
| viral vector construct (pBMN, pBABE or pMX) containing cDNA of interest | 1.5 µg |
| gag-pol vector | 1.0 µg (amount for 1 well) |
| VSV-G vector | 0.5 µg (amount for 1 well) |
| Opti-MEM (RT) | 500 µL |
Add 3µL of Plus reagent and mix well. Incubate at Room temperature for 0h 5m 0s.
Add 9µL of Lipofectamin LTX (1:3 ratio of Plus:LTX is standard in the lab but can be adjusted for your own protocol) and vortex for 0h 0m 15s. Incubate at Room temperature for 0h 20m 0s.
Once the 20 min incubation starts, replace the media in each well with 1mL DMEM/10% FBS media.
When the 20 min incubation finishes, add the optimum/liposome mix to the well.
Day 3
In the morning, remove the old media from the HEK293T cells which may contain viruses at this stage) into a beaker of beach and add 1mL of fresh growth media. The next day, viruses can be harvested for infection.
Seed the target cells (about 100k-120k cells) into a 6-well plate if intending to do infection with fresh viruses.
Day 4
In the late afternoon, collect viral supernatant from HEK293Ts, spin down at max speed for 0h 5m 0s to pellet debris and filter through 0.45µm syringe filters. Viral particles can freshly be used for infection on the cells plated out on day 3 (see below) or can be frozen at -80°C for future use.
For second harvest, add 1.5mL fresh growth media back to HEK293T cells for 2 days and harvest again (on Day 6).
For infection, harvested viruses are topped up with fresh growth media to make up a total of 2mL.
Aspirate the media from the target cells.
Add the 2mL of virus-containing media (from step 3) to the target cells. Add polybrene to a final concentration of 8µg/mL to the well and mix well.
Days 5 and 6
The viruses can be removed from the cells into a beaker of bleach after 24 h (Day 5) or 48 h (Day 6) and fresh media can be added to the wells.
All waste must be treated as viral waste for at least 3 media changes over 3 days post-infection.
