ABSTRACT
Objective:To establish a mouse macrophage line lacking NLRP3.Methods:A GFP and neomycin dual selection marker vector which contains an efficient shRNA-coding insert for mouse NLRP3,was constructed and transfected into macrophages (RAW264.7) to select the stable clone cells in G418-contained medium.Then,the expanded clone cells that retain strong GFP expression were further sorted using the popular flow cytometry.The obtained cell mix (herein termed RAWNKD) were passaged and maintained for further identification,including observation of GFP marker,especially quantitative PCR (RT-qPCR) to confirm knockdown of NLRP3 in the generated RAWNKD cells which were challenged with LPS and ATP or not.Results:Over 80% of RAWNKD cells expressed GFP,and little NLRP3 mRNA was detected in RAWNKD cells,notably no obvious increase in NLRP3 mRNA was observed when the RAWNKD cells were challenged by LPS and ATP.Conclusion:The macrophage line lacking NLRP3 was successfully established,and such macrophage deficient in NLRP3 inflammasome is a valuable cell model for investigating the activation of NLRP3 inflammasome,especially signaling in inflammation mediated by NLRP3 inflammasome.