利用CRISPR/Cas9系统构建ITGαV及ITGβ3敲除细胞系  

Establishment of ITGαV Gene and ITGβ3 Gene Knockout Cell Lines Mediated by the CRISPR/Cas9 System

作　　者：胡艺欣 吴兴一 李泽辉 靳晓慧 胡慧 HU Yixin;WU Xingyi;LI Zehui;JIN Xiaohui;HU Hui(College of Veterinary Medicine,Henan Agriculture University,Zhengzhou 450002,China;Key Laboratory for Animal-derived Food Safety of Henan Province,Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院,郑州450002 [2]河南省动物食品安全重点实验室,郑州450002

出　　处：《病毒学报》2023年第2期491-498,共8页Chinese Journal of Virology

基　　金：国家自然科学基金面上项目(项目号:31972678),题目:整合素αVβ3在猪δ冠状病毒入侵宿主细胞过程中的作用研究;国家自然科学基金(项目号:31772773),题目:猪δ冠状病毒受体筛选、鉴定以及S蛋白介导的细胞入侵机制研究;河南省高校科技创新人才支持项目(项目号:20HASTIT040),题目:整合素在猪δ冠状病毒复制过程中的功能研究。

摘　　要：本试验旨在采用CRISPR/Cas9技术获得整合素αV(IntegrinαV,ITGαV)及整合素β3(Integrinβ3,ITGβ3)基因敲除的猪睾丸(Swine testicle,ST)细胞系,进而在细胞水平上研究ITGαV及ITGβ3在猪δ冠状病毒(Porcine deltacorovirus,PDCoV)入侵过程中的作用及相互作用机制。根据GenBank上猪的ITGαV及ITGβ3基因序列分别设计三对引导RNA(sgRNA),并整合到LentiCRISPR‐V2载体上,与辅助质粒pSPA×2及pMD2.G共转染293 T细胞进行慢病毒包装,包装完成后感染ST细胞并用嘌呤霉素进行压力筛选。用T7酶进行酶切检测,选出编辑效率较高的细胞群体,进一步用有限稀释法筛选出单克隆敲除细胞系。对分离出的ST‐ITGαVKO(敲除ITGαV基因的ST细胞)及ST‐ITGβ3KO(敲除ITGβ3基因的ST细胞)的单克隆细胞系进行测序和Western Blotting鉴定,结果显示ITGαV与ITGβ3均被成功敲除。采用敲除细胞系进行PDCoV感染试验,结果表明敲除ITGαV、ITGβ3基因均可抑制PDCoV在ST细胞内的增殖,成功构建的ST‐ITGαVKO及ST‐ITGβ3KO细胞系为后续阐明ITGαV、ITGβ3在PDCoV入侵过程中的作用提供了细胞模型。We wished to establish knockout of integrin αV (ITGαV) and Integrin β3 (ITGβ3) genes in a swine testicle (ST) cell line by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) technology.Then,we aimed to study ITGαV and ITGβ3 genes in the invasion process and interaction mechanism with the porcine deltacoronavirus (PDCoV).According to the sequences of ITGαV and ITGβ3 genes for pigs in GeneBank,three pairs of guide RNAs (sgRNAs) were designed and integrated into the LentiCRISPR‐V2 vector.Then 293 cells were co‐transfected with the helper plasmid pSPA×2 and pMD2.G for lentiviral packaging.After packaging had been completed,ST cells were infected and pressure screened with puromycin.Digestion was undertaken with T7 enzyme to select cell populations with high editing efficiency.Then,we undertook limited dilutions to isolate monoclonal knockout cell lines.The isolated monoclonal cell lines of ST‐ITGαVKO(ST cells with the ITGαV gene knock‐out) and ST‐ITGβ3KO(ST cells with the ITGβ3 gene knock‐out) were identified using sequencing and western blotting.Results indicated that ITGαV and ITGβ3 genes had been knocked out successfully.A PDCoV infection assay was carried out using knockout cell lines.We discovered that knockout of the ITGαVβ3 genes could inhibite PDCoV proliferation.The present study showed that ST‐ITGαVKOand ST‐ITGβ3KOcell lines were constructed successfully.Our data could lay the foundation for elucidating the role of ITGαV and ITGβ3 in the invasion process of the PDCoV.

关 键 词：整合素ΑV 整合素Β3 CRISPR/Cas9 猪睾丸细胞 猪δ冠状病毒 

分 类 号：S828.9[农业科学—畜牧学]