中国兽医科学2025，Vol.55Issue(5)：569-576,8.DOI:10.16656/j.issn.1673-4696.2025.0067
利用CRISPR/Cas9基因编辑技术构建Nup155基因敲除的PK-15细胞系
Construction of Nup155 knockout PK-15 cell line using CRISPR/Cas9 gene editing technology
摘要
Abstract
To study the effect of Nup155 function on viral replication,CRISPR/Cas9 gene editing technology was used to construct Nup155 knockout PK-15 cell lines.Four gRNAs were designed to precisely target the Nup155 gene.The sgRNAs were synthesized and then fused with the LentiCRISPR v2 vector.The sgRNA vectors were packaged as a lentivirus in 293 cells.Cell lines were screened with 2 μg/mL purinomycin.RT-qPCR and Western-blot were used to confirm whether Nup155 gene was knocked out in cells.Finally,CCK8 assay was used to detect the effect of Nup155 gene knockout on the activity of PK-15 cells.Both RT-qPCR and Western-blot results showed that the mRNA and protein expression levels of Nup155 in single cell clones were significantly reduced.CCK8 assay results showed that PK-15 cell viability decreased in Nup155 knocked out cell lines.In conclusion,this study successfully constructed a PK-15 cell line with Nup155 partial knocked out by CRISPR/Cas9 technology,and partial knocking out Nup155 significantly affected the viability of PK-15 cells.This study provides a useful cell model for further in-depth study of Nup155 function.关键词
PK-15细胞/基因敲除/Nup155/慢病毒包装Key words
PK-15 cells/gene knockout/Nup155/lentiviral packaging分类
农业科技引用本文复制引用
孙瑜嘉,殷娟斌,刘瀛,李可卿,曹轶梅,朵红,卢曾军,赵志荀,张强..利用CRISPR/Cas9基因编辑技术构建Nup155基因敲除的PK-15细胞系[J].中国兽医科学,2025,55(5):569-576,8.基金项目
国家自然科学基金项目(32372988) （32372988）
国家重点研发计划项目(2021YFD1800300) （2021YFD1800300）
青海省重点研发与转化计划项目(2022-QY-207) （2022-QY-207）
