Apr 18, 2022
Generation of Stable STING-GFP cells using retrovirus
- Will Hancock-Cerutti1,2,3,
- Pietro De Camilli1,3
- 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Will Hancock-Cerutti, Pietro De Camilli 2022. Generation of Stable STING-GFP cells using retrovirus. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl85xp7l2w/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: July 09, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 51428
Keywords: STING, STING-GFP, Retrovirus, Retroviral transduction, ASAPCRN
Abstract
This method describes the generation of HeLa cells stably expressing STING-GFP using retroviral transduction in order to study the localization of STING under different conditions.
Attachments
dxw2bkjz7.pdf
117KB
Materials
DNA Reagents:
- STING-V1addgeneCatalog #124262
- pEGFP-N1 (Clontech)
- pMXs-IRES-Bsd Retroviral Expression VectorCell Biolabs, IncCatalog #RTV-016
The sequences for the primers used are listed in Table S1 of our manuscript.
Solutions to prepare:
DMEM (-P/S) solution:
| A | B | |
| FBS | 10% | |
| L-glutamine | 2 mM |
Safety warnings
All appropriate biosafety precautions should be observed when handling retrovirus.
Cloning of pMX-STING-GFP retroviral vector
Cloning of pMX-STING-GFP retroviral vector
2d 16h
2d 16h
Amplify the coding sequence for human STING using PrimeSTAR GXL DNA polymerase (Takara Bio) according to manufacturer protocol. Primers include a XhoI restriction site at the 5’ end and a SacII restriction site at the 3’ end.
Purify the amplicon from PCR reaction mixture using a NucleoSpin Gel and PCR Cleanup kit (Macherey-Nagel) and run amplicon in an agarose gel to confirm expected size.
Digest the hSTING PCR product and pEGFP-N1 plasmid using XhoI and SacII restriction enzymes (New England BioLabs) in CutSmart buffer (New England BioLabs) according to manufacturer protocol.
Run digested products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).
Ligate the digested hSTING amplicon and linearized pEGFP-N1 backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.
Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37 °C for 16:00:00 .
16h
Pick single bacterial colonies and expand. Grow in 5 mL LB media at 37 °C for 16:00:00 .
16h
Purify plasmid by Mini-Prep (Qiagen) and sequence.
Digest the hSTING-EGFP-N1 and pMXs-IRES-Blasticidin Retroviral Vector backbone using XhoI and NotI-HF restriction enzymes (New England BioLabs) in CutSmart buffer (New England BioLabs) according to manufacturer protocol.
Run digested products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)
Ligate the digested hSTING-EGFP amplicon and linearized pMXs-IRES-Blasticidin Retroviral Vector backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.
Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37 °C for 16:00:00 .
16h
Pick single bacterial colonies and expand. Grow in 5 mL LB media at 37 °C for 16:00:00 .
16h
Purify plasmid by Mini-Prep or Maxi-Prep (Qiagen) and sequence.
Transduction of HeLa cells with pMX-STING-GFP retrovirus
Transduction of HeLa cells with pMX-STING-GFP retrovirus
2d
2d
Plate 5 x 106 Plat-A cells (Cell Biolabs) on a 10 cm plate in DMEM (-P/S).
The following day, transfect cells with 9 µg of pMX-STING-GFP using Fugene HD (Promega).
At 48:00:00 post-transfection, plate target HeLa cells at 2.5 x 105 in DMEM (-P/S) 6 well format.
2d
At 72:00:00 post-transfection, collect retroviral supernatant into a falcon tube and supplement with 8 μg/ml Polybrene (Millipore).
3d
Pass supernatant through 0.22 μm filter to remove cellular debris and add to target HeLa cells.
At 24:00:00 post-transduction, remove retroviral supernatant and replace with fresh DMEM complete.
1d
At 48:00:00 post-transduction, sort HeLa cells by FACS to enrich for GFP positive cells.
2d