Construction of MAVS knockout ZR-751 stable strain by CRISPR/Cas9 technology / 军事医学

ABSTRACT

Objective To construct mitochondrial antiviral-signaling protein ( MAVS ) knockout ZR-751 breast neoplasms cells using CRISPR/Cas9 genome engineering technology , and study the effect of MAVS on cell proliferation . Methods Small guide RNA ( sgRNA ) was designed by targeting the first exon of MAVS gene and the pX 459-sgRNA recombinant eukaryotic expressional plasmid was constructed .Puromycin was used to screen monoclonal cells which stably knocked out MAVS gene .The knockout effect was measured by Western blotting .Cellular proliferation rates were detected by colony-forming assay when MAVS gene was knockout .The MTS assay was designed to detect the effect of MAVS on cell proliferation under DFX stimulus .Results The result of Western blotting suggested that no MAVS protein was detected in the MAVS gene knockout stable ZR-751 cells,showing that MAVS gene was knocked out completely .Proliferation became faster when MAVS was knocked out .MAVS promoted cell death under DFX stimulus .Conclusion The MAVS knockout ZR-751 stable cells have been constructed using CRISPR/Cas9 system.The preliminary experimental results show that MAVS inhibits breast cancer cell proliferation , which will facilitate studies on the function of MAVS in tumors in the future .