Generation of stable cell lines via lentiviral transduction
Elias Adriaenssens
Abstract
Here, we describe a protocol to generate stable cell lines using a lentivirus system. Please note that necessary safety measures are to be taken in working with lentivirus.
Attachments
Steps
Packaging lentiviral plasmid into a lentiviral particles for in…
Grow HEK293T cells to 60-70% confluency in Growth media in a 6-well Petri Dish.
Prepare a transfection mix in a sterile 1.5 ml Eppendorf tube, containing:
| A | B |
|---|---|
| Lentiviral vector with your gene-of-interest | 1500 ng |
| Gag/Pol plasmid | 1000 ng |
| VSV-G plasmid | 500 ng |
| P3000 reagent (Lipofectamine 3000 kit) | 5 µL |
| OptiMem | 125 µL |
Prepare Lipofectamine 3000 mixture in a sterile 1.5 ml Eppendorf tube, containing:
| A | B |
|---|---|
| Lipofectamine 3000 | 5 µL |
| OptiMem | 125 µL |
Incubate each mixture (from steps 2 and 3) separately for ~ 0h 5m 0s at Room temperature.
Mix the two suspensions and incubate at Room temperature for 0h 15m 0s.
Add the mixture drop-wise to the cells from step 1 using a P1000 sterile pipette.
Incubate cells at 37°C for 24h 0m 0s.
Collect the supernatant from the cells (that now contains the lentiviruses) and pass it through a 0.45 µm syringe filter. If needed, add fresh growth medium and collect this too 24h 0m 0s later.
Lentiviral infection of HeLa cells
Add 2µL of polybrene (8 mg/ml) to 2mL of lentivirus infection media from step 8 to HeLa cells seeded in 6-well plate (at 700.000/well) at 60% confluence.
Incubate at 37°C for 24h 0m 0s.
Change media to fresh medium and incubate until confluency. Split three times before cells can leave the viral S2 lab.
Cells can now be passaged and plated for experiments or frozen down for long-term storage in liquid nitrogen.
