Construction of RSAD2-Knockout ST Cells by CRISPR/Cas9 System
LI Juan1,2, DU Liuyang1, GU Jinyan2, CAO Ruibing1*
RSAD2 is a multifunctional IFN (interferon) inducible protein. In this study, a RSAD2 gene knockout ST (swine testicular) cell line was established using CRISPR/Cas9 system. Four sgRNAs targeting RSAD2 gene were designed using online tools and cloned into pX459 vector. Recombinant plasmids were initially screened by puromycin and detected by Western blot, confirming that RSAD2-sgRNA-4 has the best knockout effect. The study successfully obtained the RSAD2 knockout ST cell model by sub-cloned the ST cells transfected with RSAD2-sgRNA-4. Dual luciferase activity assay and RT-qPCR (real-time fluorescence quantitative PCR) test proved that RSAD2 gene knockout had no significant effect on the promoter activities and mRNA levels of IFN-β, IRF3, and NF-κB genes in ST cells. In total, the RSAD2 gene knockout ST cell model, which is constructed by CRISPR/Cas9 system, will come to be a powerful tool for studying the function of RSAD2.
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