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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0500

摘要/Abstract

摘要：

【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mCherry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。

关键词: CRISPR/Cas9技术, Pmepa1, TCMK1细胞系, 纤维化

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 Pmepa1 48 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.

Key words: CRISPR/Cas9 technique, Pmepa1, TCMK1 cell line, fibrosis

张宏民, 龙雯, 劳筱清, 陈雯妍, 商雪梅, 王洪连, 王丽, 粟宏伟, 沈宏萍, 沈宏春. 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.

ZHANG Hong-min, LONG Wen, LAO Xiao-qing, CHEN Wen-yan, SHANG Xue-mei, WANG Hong-lian, WANG Li, SU Hong-wei, SHEN Hong-ping, SHEN Hong-chun. Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2024, 40(2): 73-79.

图/表 3

图1 Pmepa1 sgRNA 靶向位置和质粒构建结果 A.小鼠Pmepa1位点基因结构和pX333-Pmepa1载体结构示意图。B. pX333-Pmepa1载体测序结果图。C. TCMK1细胞转染pX333-Pmepa1质粒48 h后的mCherry荧光图。D. TCMK1细胞转染pX333-Pmepa1质粒的流式细胞术分析图

Fig. 1 Pmepa1 sgRNA targeting location and plasmid construction results A. Schematic representation of the gene structure of the mouse Pmepa1 locus and the structure of the pX333-Pmepa1 vector. B. Sequence diagram of pX333-Pmepa1 vector. C. mCherry fluorescence plot of TCMK1 cells 48 h after transfection with pX333-Pmepa1 plasmid. D. Flow cytometry analysis plot of TCMK1 cells transfected with pX333-Pmepa1 plasmid

图2 Pmepa1基因敲除效果验证 A. 19个单克隆TCMK1细胞系Pmepa1基因Cas9作用位点邻近DNA序列PCR扩增电泳图。B. TCMK1细胞clone 4（KO-4）和clone 16（KO-16）Pmepa1基因序列突变情况分析图。C. Western blot检测Pmepa1蛋白表达

Fig. 2 Validation of Pmepa1 gene knockout A. PCR amplification electrophoresis of DNA sequences adjacent to the Cas9 action site of the Pmepa1 gene in 19 monoclonal TCMK1 cell lines. B. Sequence analysis of clone 4（KO-4）and clone 16（KO-16）PMEPA1 gene mutations in TCMK1 cells. C. Western blot for Pmepa1 protein expression

图3 RT-PCR和Western blot验证Pmepa1敲除后TGF-β信号的活化和纤维化蛋白表达结果 A. RT-PCR方法检测了纤维化相关基因表达。B. Western blot方法检测了Smad2/Smad3通路相关蛋白

Fig. 3 RT-PCR and Western blot validation of TGF-β signaling activation and fibrotic protein expression after Pmepa1 knockdown A. RT-PCR method was used to detect fibrosis-related gene expression. B. The Western blot method to detecte Smad2/Smad3 pathway-related protein. *P < 0.05, **P < 0.01, ***P < 0.001 vs +TGF-β1/- TGF-β1 group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs +TGF-β1 KO-4, KO-16/+TGF-β1 NC group

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