Sep 23, 2023

Public workspaceGeneration of stable cell lines via lentiviral transduction

DOI
dx.doi.org/10.17504/protocols.io.6qpvr3e5pvmk/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Elias Adriaenssens
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr3e5pvmk/v1
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: May 31, 2024
Protocol Integer ID: 84010
Keywords: ASAPCRN
Funders Acknowledgement:
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
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
Attention
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
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:
AB
Lentiviral vector with your gene-of-interest1500 ng
Gag/Pol plasmid1000 ng
VSV-G plasmid500 ng
P3000 reagent (Lipofectamine 3000 kit)5 µL
OptiMem125 µL
Prepare Lipofectamine 3000 mixture in a sterile 1.5 ml Eppendorf tube, containing:
AB
Lipofectamine 30005 µL
OptiMem125 µL
Incubate each mixture (from steps 2 and 3) separately for ~ Duration00:05:00 at TemperatureRoom temperature .

5m
Incubation
Mix the two suspensions and incubate at TemperatureRoom temperature for Duration00:15:00 .

15m
Incubation
Mix
Add the mixture drop-wise to the cells from step 1 using a P1000 sterile pipette.
Pipetting
Incubate cells at Temperature37 °C for Duration24:00:00 .

1d
Incubation
Collect the supernatant from the cells (that now contains the lentiviruses) and pass it through a Thikness0.45 µm syringe filter. If needed, add fresh growth medium and collect this too Duration24:00:00 later.
1d
Lentiviral infection of HeLa cells
Lentiviral infection of HeLa cells
1d
Add Amount2 µL of polybrene (8 mg/ml) to Amount2 mL of lentivirus infection media from step 8 to HeLa cells seeded in 6-well plate (at 700.000/well) at 60% confluence.

Pipetting
Incubate at Temperature37 °C for Duration24:00:00 .

1d
Incubation
Change media to fresh medium and incubate until confluency. Split three times before cells can leave the viral S2 lab.
Incubation
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.