Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department of Pathology and Immunology, Division of Immunobiology, Washington University School of Medicine, St Louis, Missouri, USA.
Kidney Int. 2017 Jun;91(6):1510-1517. doi: 10.1016/j.kint.2016.12.011. Epub 2017 Feb 7.
Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 and hNphs2.Cre mice to Phb2 mice. The podocyte-specific Phb2 knockout by Nphs2 mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.
足细胞损伤是导致蛋白尿和肾小球瘢痕形成的肾小球疾病的关键事件。因此，肾小球研究致力于发现影响足细胞健康的分子机制和信号通路。hNphs2.Cre 小鼠模型是一种用于操纵足细胞特异性基因的有价值的工具，可用于谱系追踪和纯化足细胞标记。在这里，我们设计了一种新型的足细胞特异性三顺反子 Cre 小鼠模型，该模型结合了密码子优化的 Cre 表达和 mTomato 的荧光细胞标记，由内源性 Nphs2 启动子控制，使用病毒 T2A-肽。通过免疫荧光、荧光激活细胞分选和蛋白质分析证实了内源性足突蛋白、密码子优化的 Cre 和 mTomato 的独立表达。Nphs2 小鼠正常发育，在基础条件下和疾病状态下均未显示任何肾小球疾病或脱靶效应的迹象。Nphs2 介导的基因重组优于传统的 hNphs2.Cre 小鼠介导的基因重组。最后，我们通过将 Nphs2 和 hNphs2.Cre 小鼠与 Phb2 小鼠交配，在疾病模型中比较 Cre 效率。与 hNphs2.Cre 触发的足细胞特异性 Phb2 基因缺失相比，Nphs2 小鼠的足细胞特异性 Phb2 敲除导致肾小球损伤加重。因此，我们生成了第一个结合增强的 Cre 重组酶效率和足细胞中荧光标记的三顺反子足细胞小鼠模型，而无需与传统报告小鼠系进行额外的交配。
