A plasmid encoding HSV1 UL12 M185 SPA containing a 3X FLAG tag at the C-terminus was described previously24 (link). This construct, or a plasmid encoding murine cGAS-HA (Invivogen), was subcloned into the pMXs-IRES-Puro vector and replication incompetent retroviruses were packaged using plat-E cells according to the manufacturer’s instructions (Cell Biolabs). SV40 large T immortalized cGAS−/− MEFs were exposed to supernatants containing cGAS-HA retroviruses and incubated overnight. Two days post transduction, 3 µg/ml puromycin was added to select a stable population of cells expressing cGAS-HA. Supernatants containing empty or UL12 M185 SPA retroviruses and 4 µg/ml polybrene were incubated with cells 5 × 104 MEFs or 2 × 105 BMDM in 12 well dishes for a period of 8 hours. Viral supernatants were then washed off, fresh media was added to wells, and the cells were incubated for the duration of the experiment until lysis.
Pmxs ires puro vector
Manufactured by Cell Biolabs
Sourced in United States
The PMXs-IRES-Puro vector is a mammalian expression vector that allows for the expression of two genes from a single promoter. It contains an internal ribosome entry site (IRES) sequence, which enables the translation of a second gene downstream of the IRES. The vector also includes a puromycin resistance gene, which can be used for the selection of cells expressing the vector.
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3 protocols using pmxs ires puro vector
1
Establishing Stable cGAS-HA Cell Line
2
Establishing Stable cGAS-HA Cell Line
3
Transposon Vectors for Genetic Manipulation
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To generate the transposon vectors, we first generated a pT2/shp53/shNf1/polyA vector. This vector was generated by inserting the H1 promoter‐shRNA expression cassette excised from pSUPER.retro.puro vector, and BGH‐polyA sequence PCR‐amplified from pL453 vector into pT2/Onc2 vector23 digested with HindIII and StuI. To generate pT2/shp53/shNf1/SV40‐GFP vector (NP vector), SV40‐GFP cassette was PCR‐amplified from pT2/shp53/GFP4 vector (Addgene, Cambridge, MA, USA), and inserted into pT2/shp53/shNf1/polyA vector. To generate pT2/shp53/shNf1/CMV‐PDGFA‐IRES‐DsRed vector (PNP vector), we introduced EcoRV sites upstream of the CMV promoter in pIRES2‐DsRed‐Express vector (Clontech, Mountain View, CA, USA). PDGFA was PCR‐cloned from the cDNA isolated from SW480 cell line, and inserted into pIRES2‐DsRed‐Express vector. The CMV‐PDGFA‐IRES‐DsRed sequence was excised, and inserted into pT2/shp53/shNf1/polyA vector. SB1123 was inserted into pMXs‐IRES‐Puro vector (Cell Biolabs, San Diego, CA, USA). pT2/CMV‐PDGFA‐IRES‐DsRed vector (P vector) was generated by inserting the CMV‐PDGFA‐IRES‐DsRed sequence into pT2/polyA backbone. pT2/CAG‐EGFP vector (GFP vector) was generated by inserting the CAG‐EGFP‐polyA sequence into pT2 backbone. We used the previously reported shRNA for Nf1 (5′‐GGACACAATGAGATTAGAT‐3′)24 and Trp53 (5′‐GTACATGTGTAATAGCTCC‐3′).7
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