Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology

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

Abstract

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

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.

Figures/Tables 3

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

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

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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