Slamf1 Rat shRNA Plasmid (Locus ID 498286)

Snippet: Generation of SLAMF1-silenced cells. The pGFP-V-RS construct for SLAMF1 shRNA (TG309422), a noneffective (scrambled) shRNA cassette (TR30013) as a negative control and an empty vector (TR3007) were from OriGene. Mec-1 cells were nucleofected with 3 SLAMF1-specific shRNAs (Supplemental Figure 3A) using the Cell Line V Nucleofector Kit (Lonza) and selected by puromycin (Sigma-Aldrich). SLAMF1– cells were sorted using a FACSAria III cell sorter (BD Biosciences) and cloned by serial dilution. …To generate SLAMF1 shRNA–resistant constructs, 3 synonymous mutations were introduced into the shRNA#1 targeted region of WT SLAMF1 cDNA clone, purchased from OriGene (pCMV6-Entry construct, RC223343). … SLAMF1 mutants were verified by Sanger sequencing (Supplemental Figure 5C). … The constructs were then transfected into Mec-1/SLAMF1sh generated from shRNA#1, and expression was verified by surface staining with anti-SLAMF1 antibodies, with SLAMF1 mut2 showing the highest percentages of expression (range 15%–25%) (Supplemental Figure 5D). Cells transfected with SLAMF1/mut2 were then flow sorted, RNA was extracted, and gene expression was verified by qPCR. Mock construct pCMV6-Entry (OriGene, PS100001) was used as the control