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

【目的】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,并探讨Pmepa1敲除对TGF-β刺激下TCMK1细胞纤维化的影响,为研究Pmepa1在纤维化模型中的作用提供细胞模型。【方法】根据CRISPR/Cas9设计原则设计靶向小鼠Pmepa1基因组序列的sgRNA序列,构建pX333载体并转染进入TCMK1细胞,采用流式分选m Cherry阳性细胞、单克隆细胞扩增和测序鉴定Pmepa1敲除细胞,Western blot验证Pmepa1基因敲除情况。采用RT-PCR和Western blot分析Pmepa1敲除对TGF-β1诱导的R-Smad的活化和纤维化的影响。【结果】将sgRNA设计在Pmepa1第2个外显子,pX333-Pmepa1质粒转染TCMK1细胞48 h后,观察到mCherry荧光蛋白的表达,流式细胞分析转染效率约为17%,pX333-Pmepa1质粒转染阳性细胞经流式分选和单克隆扩增培养,得到19个细胞克隆,对Pmepa1基因位点上sgRNA靶点序列PCR扩增测序分析发现2个双等位基因移码突变细胞,Western blot验证Pmepa1蛋白表达缺失,表明Pmepa1基因敲除TCMK1细胞株构建成功,与未敲除细胞相比,Pmepa1敲除细胞在TGF-β1刺激下,p-Smad2和p-Smad3的表达显著升高,并且促进纤维化基因Fibronectin和Collagen I的表达。【结论】利用CRISPR/Cas9技术成功构建Pmepa1基因敲除TCMK1小鼠肾小管上皮细胞系,为Pmepa1蛋白的功能研究建立了细胞模型以及Pmepa1在纤维化中的重要作用。

【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.