TITLE: Generation of Stable STING-GFP cells using retrovirus
AUTHORS: Will Hancock-Cerutti, Pietro De Camilli

[DESCRIPTION]
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.

[STEPS]
SECTION: Cloning of pMX-STING-GFP retroviral vector
1. 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.
SECTION: Cloning of pMX-STING-GFP retroviral vector
2. 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.
SECTION: Cloning of pMX-STING-GFP retroviral vector
3. 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.
SECTION: Cloning of pMX-STING-GFP retroviral vector
4. 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).
SECTION: Cloning of pMX-STING-GFP retroviral vector
5. Ligate the digested hSTING amplicon and linearized pEGFP-N1 backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.
SECTION: Cloning of pMX-STING-GFP retroviral vector
6. Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37 °C for 960 min.

SECTION: Cloning of pMX-STING-GFP retroviral vector
7. Pick single bacterial colonies and expand. Grow in 5 mL LB media at 37 °C for 960 min.

SECTION: Cloning of pMX-STING-GFP retroviral vector
8. Purify plasmid by Mini-Prep (Qiagen) and sequence.
SECTION: Cloning of pMX-STING-GFP retroviral vector
9. 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.
SECTION: Cloning of pMX-STING-GFP retroviral vector
10. 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)
SECTION: Cloning of pMX-STING-GFP retroviral vector
11. Ligate the digested hSTING-EGFP amplicon and linearized pMXs-IRES-Blasticidin Retroviral Vector backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.
SECTION: Cloning of pMX-STING-GFP retroviral vector
12. Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37 °C for 960 min.

SECTION: Cloning of pMX-STING-GFP retroviral vector
13. Pick single bacterial colonies and expand. Grow in 5 mL LB media at 37 °C for 960 min.

SECTION: Cloning of pMX-STING-GFP retroviral vector
14. Purify plasmid by Mini-Prep or Maxi-Prep (Qiagen) and sequence.
SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
15. Plate 5 x 106 Plat-A cells (Cell Biolabs) on a 10 cm plate in DMEM (-P/S).
SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
16. The following day, transfect cells with 9 µg of pMX-STING-GFP using Fugene HD (Promega).

SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
17. At 2880 min post-transfection, plate target HeLa cells at 2.5 x 105 in DMEM (-P/S) 6 well format.

SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
18. At 4320 min post-transfection, collect retroviral supernatant into a falcon tube and supplement with 8 μg/ml Polybrene (Millipore).

SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
19. Pass supernatant through 0.22 μm filter to remove cellular debris and add to target HeLa cells.

SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
20. At 1440 min post-transduction, remove retroviral supernatant and replace with fresh DMEM complete.

SECTION: Transduction of HeLa cells with pMX-STING-GFP retrovirus
21. At 2880 min post-transduction, sort HeLa cells by FACS to enrich for GFP positive cells.