中国兽医科学2025，Vol.55Issue(12)：1591-1598,8.DOI:10.16656/j.issn.1673-4696.2025.0222
CRISPR/Cas9介导的JMJD6基因敲除细胞系的建立及其应用
Establishment and application of the cell line with the JMJD6 gene knockout mediated by CRISPR/Cas9
摘要
Abstract
To investigate the influence of the JMJD6 gene on the replication of the foot-and-mouth disease virus(FMDV),CRISPR/Cas9 gene editing technology was employed to design and construct a cell line with the JMJD6 gene knocked out.Subsequently,FMDV infection tests were conducted in these cells to comprehensively analyze the role of the JMJD6 gene in FMDV proliferation.In this study,two single guide RNAs(sgRNAs)targeting the porcine JMJD6 gene were designed and ligated with the pX459 vector using T4 ligase.The recombinant plasmids were then transfected into the PK-15 porcine kidney cell line.Puromycin served as a selection agent to identify cells with the JMJD6 gene knocked out,and then the limiting dilution method was employed to obtain monoclonal cell strains.DNA sequencing and Western-blot were performed on these monoclonal cell strains to confirm the effectiveness of the JMJD6 gene knockout.The results of the FMDV infection tests indicated that,compared to the PK-WT cells,the PK-JMJD6-KO cell line exhibited a significant reduction in the protein levels,gene expression,and virus titer of FMDV.These results indicated that the CRISPR/Cas9 gene editing technology was success-fully utilized to create the PK-15 cell line with the JMJD6 gene knocked out,confirming the critical role of the JMJD6 gene in FMDV proliferation and establishing a foundation for further research into the regulatory mechanisms by which the JMJD6 gene promotes FMDV proliferation.关键词
口蹄疫病毒/JMJD6基因/CRISPR/Cas9/PK-15细胞Key words
foot-and-mouth disease virus/JMJD6 gene/CRISPR/Cas9/PK-15 cell分类
农业科技引用本文复制引用
周婷婷,蔡建涛,曹伟军,蒲秀瑛,张伟,郑海学,杨帆,杨洋,齐晓兰,邵文华,黄梦瑶,董星艳,陈治彤,刘泓屹..CRISPR/Cas9介导的JMJD6基因敲除细胞系的建立及其应用[J].中国兽医科学,2025,55(12):1591-1598,8.基金项目
国家自然科学基金项目(32473064) （32473064）
国家重点研发计划项目(2021YFD1800302) （2021YFD1800302）
兰州大学中央高校基本科研业务费专项资金(lzujbky-2022-ey20) （lzujbky-2022-ey20）
国家生猪产业技术体系项目(CARS-35) （CARS-35）
国家生猪技术创新中心项目(NC-TIP-XD/C03) （NC-TIP-XD/C03）
甘肃省自然科学基金项目(23JRRA547) （23JRRA547）
