[Construction of mouse podocyte clone-5 cell lines with <i>Smad3</i> knockout by CRISPR/Cas9].

This study established the mouse podocyte clone-5 (MPC5) with <i>Smad3</i> knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with <i>Smad3</i> knockout, aiming to provide a cell tool for studying the role of <i>Smad3</i> in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting <i>Smad3</i> 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 <i>Smad3</i> and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of <i>Smad3</i>. Finally, the effect of <i>Smad3</i> 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 <i>Smad3</i>. 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 <i>Smad3</i> 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 <i>Smad3</i> knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes <i>fibronectin</i> and <i>Col1a1</i> (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 <i>Smad3</i> knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with <i>Smad3</i> 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.