生物技术通报2024，Vol.40Issue(2)：73-79,7.DOI:10.13560/j.cnki.biotech.bull.1985.2023-0500
利用CRISPR/Cas9技术构建Pmepa1基因敲除的TCMK1小鼠肾小管上皮细胞系
Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology
摘要
Abstract
[Objective]CRISPR/Cas9 technology was used to construct Pmepa1 knockdown TCMK1 mouse renal tubular epithelial cell lines and to explore the effect of Pmepa1 knockdown on TGF-β-stimulated TCMK1 cell fibrosis,which provides a cell model for studying the role of Pmepa1 in fibrosis models.[Method]The pX333 vector was created and transfected into TCMK1 cells in accordance with the CRISPR/Cas9 design principle.Flow sorting of mCherry-positive cells,monoclonal cell expansion,and sequencing were used to identify the Pmepa1 knockout cells,and Western blot was used to confirm the knockout status of the Pmepa1 gene.Pmepa1 knockdown was confirmed using a Western blot.By using RT-PCR and Western blot,the effects of Pmepa1 knockdown on TGF-1-induced R-Smad activation and fibrosis were examined.[Result]The sgRNA was designed in exon 2 of Pmepa148 h after transfection of TCMK1 cells with pX333-Pmepa1 plasmid,the expression of mCherry fluorescent protein was observed,and flow cytometric analysis of the transfection efficiency was about 17%.pX333-Pmepa1 plasmid transfection-positive cells were cultured by flow sorting and monoclonal amplification to obtain 19 cell clones.PCR amplification sequencing analysis of the sgRNA target sequence at the PMEAP1 gene locus revealed 2 double allele shift mutant cells,and Western blot verified the absence of Pmepa1 protein expression,indicating that the Pmepa1 knockdown TCMK1 cell line was successfully constructed,compared with the non-knockdown cells,the Pmepa1 knockdown cells were stimulated by TGF-β,p-Smad2 and p-Smad3 expression was significantly elevated in Pmepa1 knockout cells compared with non-knockout cells,and the expressions of the fibrogenic genes Fibronectin and Collagen I were promoted.[Conclusion]CRISPR/Cas9 technique was used to construct the renal tubular epithelial cell line of PMEPA1 knockout TCMK1 mice successfully,which established the cell model for the functional study of PMEPA1 protein and the important role of PMEPA1 in fibrosis.关键词
CRISPR/Cas9技术/Pmepa1/TCMK1细胞系/纤维化Key words
CRISPR/Cas9 technique/Pmepa1/TCMK1 cell line/fibrosis引用本文复制引用
张宏民,沈宏春,龙雯,劳筱清,陈雯妍,商雪梅,王洪连,王丽,粟宏伟,沈宏萍..利用CRISPR/Cas9技术构建Pmepa1基因敲除的TCMK1小鼠肾小管上皮细胞系[J].生物技术通报,2024,40(2):73-79,7.基金项目
四川省科技计划资助(2022YFS0621,2022YFS0635) （2022YFS0621,2022YFS0635）
