Septins are a conserved, essential family of GTPases that interact with actin, microtubules, and membranes and form scaffolds and diffusion barriers in cells. Several of the 13 known mammalian septins assemble into nonpolar, multimeric complexes that can further polymerize into filamentous structures. While some GFP‐coupled septins have been described, overexpression of GFP‐tagged septins often leads to artifacts in localization and function. To overcome this ubiquitous problem, we have here generated a genome‐edited rat fibroblast cell line expressing Septin 2 (Sept2) coupled to enhanced green fluorescent protein (EGFP) from both chromosomal loci. We characterize these cells by genomic polymerase chain reaction (PCR) for genomic integration, by western blot and reverse transcriptase‐PCR for expression, by immunofluorescence and immunoprecipitation for the colocalization of septins with one another and cellular structures and for complex formation of different septins. By live cell imaging, proliferation and migration assays we investigate proper function of septins in these cells. We find that EGFP is incorporated into both chromosomal loci and only EGFP‐coupled Sept2 is expressed in homozygous cells. We find that endogenous Sept2‐EGFP exhibits expression levels, localization and incorporation into cellular septin complexes similar to the wt in these cells. The expression level of other septins is not perturbed and cell division and cell migration proceed normally. We expect our cell line to be a useful tool for the cell biology of septins, especially for quantitative biology.

中文翻译：

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纯合基因组编辑的Sept2-EGFP成纤维细胞系。

Septins是GTPases的一个必不可少的保守家族，可与肌动蛋白，微管和膜相互作用，并在细胞中形成支架和扩散屏障。13种已知的哺乳动物Septin中有几种组装成非极性的多聚体复合物，可以进一步聚合成丝状结构。虽然已经描述了一些GFP偶联的Septin，但GFP标记的Septin的过表达通常会导致定位和功能方面的伪影。为了克服这个普遍存在的问题，我们在这里生成了一个基因组编辑的大鼠成纤维细胞，该细胞表达Septin 2（Sept2），并与来自两个染色体位点的增强型绿色荧光蛋白（EGFP）偶联。我们通过基因组聚合酶链反应（PCR）进行基因组整合，Western印迹和逆转录酶-PCR进行表达来表征这些细胞，通过免疫荧光法和免疫沉淀法使隔蛋白与细胞结构彼此共定位，并形成不同的隔蛋白的复合物。通过活细胞成像，增殖和迁移分析，我们研究了这些细胞中隔蛋白的适当功能。我们发现EGFP已整合到两个染色体位点中，并且纯合细胞中仅表达了EGFP偶联的Sept2。我们发现内源性Sept2-EGFP的表达水平，定位和掺入细胞Septin复合物的过程类似于 我们发现EGFP已整合到两个染色体位点中，并且纯合细胞中仅表达了EGFP偶联的Sept2。我们发现内源性Sept2-EGFP的表达水平，定位和掺入细胞Septin复合物的过程类似于 我们发现EGFP已整合到两个染色体位点中，并且纯合细胞中仅表达了EGFP偶联的Sept2。我们发现内源性Sept2-EGFP的表达水平，定位和掺入细胞Septin复合物的过程类似于这些细胞中的wt。其他Septins的表达水平不受影响，细胞分裂和细胞迁移正常进行。我们希望我们的细胞系将成为Septins细胞生物学的有用工具，尤其是定量生物学。