Generation of stable cell lines via retroviral or lentiviral transduction
Dario R Alessi, Rotimi Fasimoye
Abstract
The ability to stably express a protein of interest in cells is critical to study its function. Here, we describe a protocol to generate stable cell lines using a retrovirus system that can be used for a variety of mouse and human cell lines. Our protocol includes the production of retroviruses encoding the transgene of interest in HEK293FT and the subsequent transduction of the cell line that is intended to stably express the protein of interest. The same protocol can also be used to generate stable cell lines using a lentivirus system. It should be noted that when using this method, the transgene of interest will be randomly integrated into the cell genome.
Attachments
Steps
Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
10 cm Petri Dish. Prepare a transfection mix in a sterile 1.5 ml Eppendorf tube, containing:
3.8µgpGag/Pol plasmid2.2µgpVSVG plasmid6µgpBABE-SLC35A2-2xFLAG plasmid300µLOptiMem
Prepare PEI mixture in a sterile 1.5 ml Eppendorf tube, containing:
20µL1mg/mLPEI Max 40K dissolved in distilled water.300µLOptiMem.
Incubate each mixture (from steps 2 and 3) separately for ~0h 5m 0s at Room temperature, then combine.
Mix by vortexing and incubate at Room temperature for 0h 30m 0s.
Add the mixture dropwise to the cells from step 2 using a P1000 sterile pipette.
Incubate cells at 37°C for 24h 0m 0s.
Replace media with 10mL of fresh Growth Media and incubate cells for a further 24h 0m 0s at 37°C.
Collect the culture media from step 8 (that now contains the retroviruses) and pass through a 0.45 µm syringe filter.
Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
Mix 5mL of retrovirus infection media from step 9 with 5mL of fresh Growth Media in a sterile 15 ml Eppendorf tube.
Add Polybrene (10mg/mL stock dissolved in MilliQ water, sterile filtered) to a final concentration of 10µg/ml.
Gently add to a 10 cm plate of HEK293 cells (or any cell line of interest) at ~60% confluency.
Incubate at 37°C for 24h 0m 0s.
Change media to Growth Media and incubate for another 24h 0m 0s at 37°C.
To select cells stably expressing SLC35A2-2xFLAG, replace media with 10mL of freshly prepared Selection Media.
Change Selection Media every 24 hr for 72h 0m 0s - 120h 0m 0s to remove dead cells. After 5 days, cells stably expressing SLC35A2-2xFLAG should have reached 100% confluency.
Cells can now be passaged and plated for experiments, or frozen down for long term storage in liquid nitrogen (Freezing media: growth media added with 10% v/v DMSO).
