Apr 15, 2023

Public workspaceGeneration of stable cell lines using retroviral system

DOI
dx.doi.org/10.17504/protocols.io.81wgbyez1vpk/v1
  • 1Laboratory of Michael Lazarou, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
nguyen.tha
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DOI: dx.doi.org/10.17504/protocols.io.81wgbyez1vpk/v1
Protocol Citation: nguyen.tha 2023. Generation of stable cell lines using retroviral system. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyez1vpk/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: April 10, 2023
Last Modified: April 15, 2023
Protocol Integer ID: 80233
Keywords: stable cell lines, retroviral system
Abstract
This protocol details generation of stable cell lines using retroviral system.
Attachments
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698-1486.docx
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Guidelines
Attention

  • The HEK293T cells detach very easily, be extra gentle when changing the media.

Materials
Buffers and reagents:

  • Polybrene (4 mg/mL)

Growth media:
AB
DMEM with 10% FBS
Glucose4.5 g/l
GlutaMAXTM1x
MEM NEAA1x
HEPES25 mM
Reagent45% D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8769

ReagentGlutaMAX™ SupplementThermo FisherCatalog #35050061

ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140050

ReagentHEPES Buffer 1M Solution Cell Culture Grade MP BiomedicalsFisher ScientificCatalog #ICN1688449

ReagentLipofectamine™ LTX Reagent with PLUS™ ReagentThermo FisherCatalog #A12621

ReagentGibco™ Opti-MEM™ I Reduced Serum Medium no phenol redFisher ScientificCatalog #11-058-021

ReagentMillex-HV Syringe Filter Unit 0.45 µm PVDF 33 mm gamma-sterilizable sterilizedMerck MilliporeSigma (Sigma-Aldrich)Catalog #SLHVM33RS


Safety warnings
Attention

  • All viral waste must be bleached and left under UV light for at least 30’ after viral work in TC hoods before disposal.
Day 1
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).
Note
Set up 1 well for each construct you wish to generate a virus harvest for, can be scaled up according to your need.

Day 2: The following protocol is designed for one well of the 6-well plate
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:
AB
viral vector construct (pBMN, pBABE or pMX) containing cDNA of interest1.5 µg
gag-pol vector1.0 µg (amount for 1 well)
VSV-G vector0.5 µg (amount for 1 well)
Opti-MEM (RT)500 µL
Add Amount3 µL of Plus reagent and mix well. Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Pipetting
Mix
Add Amount9 µL 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 Duration00:00:15 . Incubate at TemperatureRoom temperature for Duration00:20:00 .

20m 15s
Incubation
Pipetting
Once the 20 min incubation starts, replace the media in each well with Amount1 mL DMEM/10% FBS media.
When the 20 min incubation finishes, add the optimum/liposome mix to the well.
Note
Do it gently on the side of the well.

Day 3
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 Amount1 mL of fresh growth media. The next day, viruses can be harvested for infection.

Pipetting
Seed the target cells (about 100k-120k cells) into a 6-well plate if intending to do infection with fresh viruses.
Day 4
Day 4
In the late afternoon, collect viral supernatant from HEK293Ts, spin down at max speed for Duration00:05:00 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 Temperature-80 °C for future use.

5m
For second harvest, add Amount1.5 mL fresh growth media back to HEK293T cells for 2 days and harvest again (on Day 6).
Pipetting
For infection, harvested viruses are topped up with fresh growth media to make up a total of Amount2 mL .
Aspirate the media from the target cells.
Add the Amount2 mL of virus-containing media (from step 3) to the target cells. Add polybrene to a final concentration of Amount8 µg/mL to the well and mix well.
Pipetting
Mix
Days 5 and 6
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.