Construction and identification of 3D4/21 cell line with SIRT3 gene knockout based on CRISPR/Cas9 technology
[Objective]The aim is to construct a porcine alveolar macrophage cell line with stable knockdown of SIRT3 gene and analyze the regulatory effect of SIRT3 knockdown on virus-induced inflammatory response,thus laying the experimental foundation for an in-depth study of the role of SIRT3 gene in host antiviral infection.[Method]In this study,CRISPR/Cas9 gene editing technology was used to design sgRNA against exon 2 of porcine SIRT3 gene and transfected 3D4/21 cells after ligating pb-U6-puro-BFP plasmid.The monoclonal cells was screened,and the monoclonal cells were identified by combining Sanger sequencing,qPCR,and Western blot,and then obtained the porcine alveolar macrophage cell line with knockout of SIRT3 gene(3D4/21-SIRT3-KO).Influenza virus A/WSN/33 was used to infect wild-type and 3D4/21-SIRT3-KO porcine alveolar macrophages,and the expression levels of inflammation-associated factors IL-6 and IL-8 were detected by qPCR to preliminarily analyze the role of the SIRT3 gene in the inflammatory response induced by influenza virus infection.[Result]Sanger sequencing results showed that in the porcine alveolar macrophage clones selected to obtain knockout of the SIRT3 gene,a base deletion was generated at exon 2 locus of the SIRT3 gene and resulted in a shifted-code mutation;Meanwhile,the expression of SIRT3 mRNA and protein levels in porcine alveolar macrophages were detected using qPCR and Western blot,and the results showed that neither SIRT3 mRNA and SIRT3 protein was expressed in 3D4/21-SIRT3-KO cells compared with wild-type cells.Influenza virus infection of SIRT3 knockout porcine alveolar macrophages revealed that knockout of SIRT3 significantly exacerbated the inflammatory response induced by influenza virus infection.[Conclusion]In this study,a SIRT3 knockout porcine alveolar macrophage cell line was successfully constructed using CRISPR/Cas9 gene editing technology and preliminarily analyzed the role of SIRT3 in influenza virus infection.
国家科技期刊平台
NSTL
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