Generation of hSTING-N154S transgenic lines

Transgene generation involved inserting the hSTING-N154S mutant cDNA downstream of the murine Vav1 gene promoter to obtain hematopoietic cell-specific expression. The 2.3/4.4(HS21/45) Vav-hCD4 (Clone#2) 11.2-Kb vector generously provided by Dr. Jerry Adams (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) (Ogilvy et al, 1999) was used (Fig S7B). The Vav1-hCD4 construct was digested with SfiI and NotI to remove the hCD4 cDNA, and this was replaced with a synthetic (Celtek) cDNA encoding the consensus hSTING sequence plus the N154S point mutation reported in Liu et al (2014). The SfiI site within the mutant STING cDNA was eliminated via codon substitution to facilitate cloning of the cDNA into the Vav1 backbone using a SfiI/NotI digestion (Fig S7C). Bacteria carrying the plasmid were grown in Luria broth (244620; BD Difco) with 100 μg/ml ampicillin (A9518; Sigma-Aldrich) and purified with the PureLink HiPure Plasmid Midiprep Kit (K210015; Invitrogen). The DNA fragment containing the transgene (Fig S7B) was removed from the vector by HindIII digestion and purified using Promega’s Wizard SV Gel and PCR Clean-Up System A9281. Transgenic lines were produced via pronuclear microinjection of the Vav1 gene promoter-hSTING-N154S construct into C57BL/6 × DBA F1 embryos at the University of Calgary’s Clara Christie Centre for Mouse Genomics. Founders were identified using the following primers: SOEcolchF 5′-GGC GGT GGT GAA GGA ACG AG-3′ and SOEcolchR 5′-CCT TGA TGC CAG CAC GGT CA-3′, 5% DMSO with a cycling program of 95°C 3 min (95°C 15 s, 69°C 15 s, and 72°C 60 s) × 35 s, 72°C 5 min, using the KAPA (D-MARK KK7352) Hot Start genotyping system. Five hSTING-N154S founders were identified and three of these that showed paw swelling had been backcrossed onto the C57BL/6 background for between five and eight generations during these experiments. All transgenic mice used were hemizygous for the transgene.