利用CRISPR/Cas9技术构建Smad3基因敲除MPC5细胞系

Construction of mouse podocyte clone-5 cell lines with Smad3 knockout by CRISPR/Cas9

摘要:

本研究旨在利用CRISPR/Cas9构建Smad3基因敲除的MPC5小鼠足细胞系细胞模型，并探讨TGF-β1刺激对Smad3基因敲除后MPC5细胞去分化的影响，为Smad3在小鼠足细胞中的功能研究提供工具细胞。根据CRISPR/Cas9设计原理设计针对靶向小鼠Smad3基因单向导sgRNA序列，将连接后的pX458-Smad3载体转化至感受态细胞中，提取质粒并转染MPC5细胞。将转染后的细胞采用流式细胞分选仪分选出转染成功的细胞，经单克隆细胞扩增后采用PCR扩增Smad3基因敲除靶点附近序列并测序，分析筛选出潜在敲除细胞并在蛋白水平上验证Smad3蛋白缺失情况来确定敲除效果。在RNA水平和蛋白水平上采用逆转录-聚合酶链反应(reverse transcription-polymerase chain reacting,RT-PCR)和Western blotting、细胞免疫荧光检测分析Smad3敲除对TGF-β1诱导的MPC5的去分化的影响。根据CRISPR/Cas9设计原理，sgRNA最终设计在Smad3第5个外显子。成功构建pX458-Smad3质粒，并将质粒转染MPC5细胞24 h后，根据EGFP荧光蛋白的表达情况确定质粒转染是否成功，将转染成功的细胞经流式细胞分析转染效率，成功率约为0.1%。采用流式细胞分选仪筛选出带有绿色荧光标签转染成功的细胞，经单克隆扩增培养，最终可以得到21个细胞克隆。对Smad3基因位点上sgRNA靶点附近序列PCR扩增测序分析，发现了2个双等位基因移码突变细胞克隆，在蛋白水平上验证其Smad3蛋白表达缺失，表明成功获得2株Smad3基因敲除MPC5细胞株。在正常MPC5细胞中，TGF-β1刺激促进纤维化基因fibronectin和Col1a1(collagen I) mRNA和蛋白的表达，同时抑制足细胞分子标记蛋白synaptopodin和podocin的表达，提示足细胞的上皮-间质转分化。然而，在2株Smad3敲除MPC5细胞中，TGF-β1诱导上皮-间质转分化基因表达特征被显著抑制。本研究成功构建了2株Smad3基因敲除MPC5小鼠足细胞系，为进一步研究Smad3蛋白的功能建立了细胞模型，为研究Smad3在MPC5细胞去分化中的作用提供了思路。

Abstract:

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