[Construction of mouse podocyte clone-5 cell lines with knockout by CRISPR/Cas9].

This study established the mouse podocyte clone-5 (MPC5) with knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with knockout, aiming to provide a cell tool for studying the role of in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of . Finally, the effect of knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of . EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes and (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.