Knockout (KO) or exogenous expression of a gene of interest in cultured cells is one of the most important ways to study the function of the gene. Compared with transient transfection, stable cell lines possess great advantages such as excellent cell homogeneity and feasibility for long-term use. However, technical challenges in generating stable cell lines still exist in many laboratories using conventional techniques like limiting dilution or cloning cylinders. In this study we describe an optimized method to efficiently create stable cell lines for functional studies. This method was successfully used to generate a PIEZO1 gene-KO cell line with the CRISPR/Cas9 technology, and TRPC5/GCaMP6f-mCherry-coexpressing cell lines without antibiotic selection. Monoclonal cell lines can be obtained in 2–4 weeks after transfection. This method does not require any special equipment or consumables and can be conducted in all laboratories with general cell-culture facility.

中文翻译：

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一种简单有效的方法来产生基因敲除和转基因细胞系

基因敲除（KO）或目的基因在培养细胞中的外源表达是研究基因功能的最重要方法之一。与瞬时转染相比，稳定的细胞系具有巨大的优势，例如出色的细胞均质性和长期使用的可行性。但是，在许多实验室中，使用常规技术（如限制稀释或克隆量瓶）仍存在产生稳定细胞系的技术难题。在这项研究中，我们描述了一种优化的方法，可以有效地创建用于功能研究的稳定细胞系。此方法已成功用于生成PIEZO1使用CRISPR / Cas9技术的基因-KO细胞系以及未选择抗生素的TRPC5 / GCaMP6f-mCherry共表达细胞系。转染后2-4周即可获得单克隆细胞系。该方法不需要任何专用设备或消耗品，并且可以在具有常规细胞培养设施的所有实验室中进行。