利用CRISPR/Cas9技术构建ACBD3基因敲除Hela细胞系  

Construction of Hela Cell Lines with ACBD3 Gene Knockout Mediated by CRISPR/Cas9 Technology

作　　者：冯晓楠 贾天军[1] FENG Xiao-nan;JIA Tian-jun(Key Laboratory of Clinicial Laboratory Diagnosis,Hebei North University,Zhangjiakou,Hebei,075000,China)

机构地区：[1]河北北方学院临床检验诊断学重点实验室,河北张家口075000

出　　处：《动物医学进展》2024年第12期32-38,共7页Progress In Veterinary Medicine

基　　金：河北省自然科学基金项目(C2020405014)。

摘　　要：利用CRISPR/Cas9技术敲除Hela细胞的乙酰辅酶A结合结构域3(ACBD3),构建ACBD3基因敲除的Hela细胞系,便于后续研究ACBD3对肺炎衣原体(Chlamydia pneumoniae)生长发育的影响及相关信号通路。根据CRISPR/Cas9设计原则,设计向导RNA(sgRNA),将sgRNA与Cas9蛋白即RNP复合物通过电转染至Hela细胞,挑取单克隆细胞进行培养鉴定,利用Western blot和免疫荧光法检测ACBD3蛋白表达情况。测序显示成功构建敲除细胞株,Western blot和免疫荧光结果显示ACBD3蛋白不表达。利用CRISPR/Cas9技术成功构建ACBD3基因敲除的Hela细胞系,为进一步研究该基因对Cpn生长发育的作用及相关信号通路奠定了基础。The purpose of this study was to use CRISPR/Cas9 technique to knock out the acyl-CoA binding domain containing 3(ACBD3)in Hela cells and construct Hela cell line with ACBD3 gene knockout,so as to facilitate subsequent research on the effects of ACBD3 on the growth and development of Chlamydia pneumoniae(Cpn)and related signaling pathways.According to the CRISPR/Cas9 design principles,guide RNA(sgRNA)was designed,and the complex of sgRNA and Cas9 proteins(RNP)were transfected into Hela cells by electrotransfection.Monoclonal cells were selected for culture identification,and the expression of ACBD3 protein was detected by Western blot and immunofluorescence.Sequencing showed successful construction of knockout cell lines,and Western blot and immunofluorescence results showed that ACBD3 protein was not expressed.The ACBD3 knockout cell line was successfully constructed using CRISPR/Cas9 technology,which laid a foundation for further study of the role of this gene on the growth and development of Cpn and related signaling pathways.

关 键 词：CRISPR/Cas9技术 ACBD3基因 HELA细胞 基因敲除 肺炎衣原体 

分 类 号：Q789[生物学—分子生物学]