Sep 24, 2023
Generation of stable cell lines via lentiviral transduction
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Elias Adriaenssens 2023. Generation of stable cell lines via lentiviral transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3e5pvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 26, 2023
Last Modified: June 01, 2024
Protocol Integer ID: 84010
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
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
751-1919.pdf
120KB
Materials
MATERIALS
Cell lines
- HEK293T for virus packaging and propagation
- HeLa cells
Plasmids
- Gag/Pol plasmid
- VSV-G plasmid
- Lentiviral vectors (pHAGE-FKBP-GFP-GOI)
Note
Note: We purify plasmids using a QIAGEN Plasmid Maxi kit following the manufacturer’s protocols and ensure sterile reagents are used and mixtures prepared in tissue culture hood to avoid contamination.
Media and Reagents
- DMEM
- 10% FBS
- 1% Penicillin-Streptomycin
- 1% non-essential amino acids
- 25 mM HEPES
Transfection media (for HEK293T cells)
- Opti-MEM I Reduced Serum Medium (Gibco)
- Lipofectamine 3000 (ThermoFisher) or PEI Max (MW 40000, Polysciences)
- Polybrene (Sigma)
Safety warnings
Please note that necessary safety measures must be taken to work with lentivirus.
Packaging lentiviral plasmid into a lentiviral particles for in…
Packaging lentiviral plasmid into a lentiviral particles for in…
2d 0h 20m
2d 0h 20m
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 ~ 00:05:00 at Room temperature .
5m
Mix the two suspensions and incubate at Room temperature for 00:15:00 .
15m
Add the mixture drop-wise to the cells from step 1 using a P1000 sterile pipette.
Incubate cells at 37 °C for 24:00:00 .
1d
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 24:00:00 later.
1d
Lentiviral infection of HeLa cells
Lentiviral infection of HeLa cells
1d
1d
Add 2 µL of polybrene (8 mg/ml) to 2 mL 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 24:00:00 .
1d
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.
Note
Freezing media: growth media added with 20% FBS and 10% v/v DMSO.