基于CRISPR/Cas9技术的SIRT3基因敲除猪肺泡巨噬细胞系的构建与鉴定
Construction and identification of 3D4/21 cell line with SIRT3 gene knockout based on CRISPR/Cas9 technology
王宝鑫 1张文华 1董霞 1郑好 1张晶 1陈洪波 1周傲1
作者信息
- 1. 武汉轻工大学 动物科学与营养工程学院/动物遗传繁育与精准养殖实验室,湖北 武汉 430023;湖北省家畜种业技术创新中心,湖北 武汉 430023
- 折叠
摘要
[目的]旨在构建稳定敲除SIRT3基因的猪肺泡巨噬细胞系,分析SIRT3敲除对病毒诱导的炎症反应的调控作用,为探究SIRT3基因在宿主抗病毒感染中的作用奠定试验基础.[方法]研究利用CRISPR/Cas9基因编辑技术,针对猪SIRT3基因第2号外显子设计sgRNA,连接pb-U6-puro-BFP质粒后转染3D4/21细胞,筛选单克隆细胞,结合Sanger测序、qPCR和Western blot技术对获得的单克隆细胞进行鉴定,进而获得SIRT3基因敲除的猪肺泡巨噬细胞系(3D4/21-SIRT3-KO),并以流感病毒A/WSN/33为研究对象,感染野生型和3D4/21-SIRT3-KO猪肺泡巨噬细胞,利用qPCR方法检测炎症相关因子IL-6、IL-8的表达水平,初步分析SIRT3基因在流感病毒感染诱导炎症反应的影响.[结果]Sanger测序结果显示,在挑选获得的敲除SIRT3基因的猪肺泡巨噬细胞克隆中,SIRT3基因第2号外显子位点产生了碱基缺失,并导致移码突变;同时,利用qPCR和Western blot检测猪肺泡巨噬细胞中SIRT3 mRNA和蛋白水平的表达,结果显示与野生型细胞相比,3D4/21-SIRT3-KO细胞中SIRT3 mRNA和SIRT3蛋白均不表达;流感病毒感染SIRT3基因敲除的猪肺泡巨噬细胞时发现,敲除SIRT3能显著加剧流感病毒感染诱导的炎症反应.[结论]研究利用CRISPR/Cas9基因编辑技术成功构建了SIRT3基因敲除的猪肺泡巨噬细胞系,并初步分析了SIRT3在流感病毒感染中作用.
Abstract
[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.
关键词
猪肺泡巨噬细胞系/SIRT3/CRISPR/Cas9/炎症反应/抗病育种/基因编辑Key words
SIRT3/CRISPR/Cas9/inflammatory reaction/disease-resistance breeding/gene editing引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据