Generation of epitope tag knock-in mice with CRISPR-Cas9 to study the function of endogenous proteins.
Generation of epitope tag knock-in mice with CRISPR-Cas9 to study the function of endogenous proteins.
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DOI：
10.1016/j.xpro.2023.102518
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发表时间：
2023-09-15
期刊：
影响因子：
--
通讯作者：
Zhang, Zhao
中科院分区：
文献类型：
--
作者：
Zhang, Zhao
研究小鼠内源性蛋白质提供了许多见解，这些蛋白质的生理和病理作用。然而，蛋白质特异性抗体的可用性和特异性往往限制了此类研究。在这里，我们提出了一种利用CRISPR-Cas9介导的基因编辑产生表位标签敲入小鼠的方案。我们讨论了标签选择和敲入位置的关键考虑因素，并提供了CRISPR设计的见解。随后，我们概述了在敲入小鼠一代，基因分型，和验证，并探讨潜在的应用程序所涉及的顺序步骤。有关本方案使用和执行的完整详细信息，请参见Zhang et al.（2022年）。使用CRISPR-Cas9有效生成表位标签敲入小鼠的方案标签选择、引物设计和质粒构建的详细讨论使用Surveyor分析测试CRISPR-Cas9在细胞系中的效率表位标签敲入小鼠生成和验证的分步指南出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。研究小鼠内源性蛋白质提供了许多见解，这些蛋白质的生理和病理作用。然而，蛋白质特异性抗体的可用性和特异性往往限制了此类研究。在这里，我们提出了一种利用CRISPR-Cas9介导的基因编辑产生表位标签敲入小鼠的方案。我们讨论了标签选择和敲入位置的关键考虑因素，并提供了CRISPR设计的见解。随后，我们概述了在敲入小鼠一代，基因分型，和验证，并探讨潜在的应用程序所涉及的顺序步骤。
Studying endogenous proteins in mice has provided numerous insights into the physiological and pathological roles of these proteins. However, the availability and specificity of protein-specific antibodies often limit such studies. Here we present a protocol for generating epitope tag knock-in mice with CRISPR-Cas9-mediated gene editing. We discuss key considerations for tag selection and knock-in location and provide insights into CRISPR design. Subsequently, we outline the sequential steps involved in knock-in mouse generation, genotyping, and validation and explore potential applications. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022). Protocol for efficiently generating epitope tag knock-in mice using CRISPR-Cas9 Detailed discussion of tag selection, primer design, and plasmid construction Testing the efficiency of CRISPR-Cas9 in cell lines with a surveyor assay Step-by-step guide for epitope tag knock-in mouse generation and validation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Studying endogenous proteins in mice has provided numerous insights into the physiological and pathological roles of these proteins. However, the availability and specificity of protein-specific antibodies often limit such studies. Here we present a protocol for generating epitope tag knock-in mice with CRISPR-Cas9-mediated gene editing. We discuss key considerations for tag selection and knock-in location and provide insights into CRISPR design. Subsequently, we outline the sequential steps involved in knock-in mouse generation, genotyping, and validation and explore potential applications.
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影响因子：
16.6
作者：
Zhang, Zhao;Xun, Yu;Rong, Shunxing;Yan, Lijuan;SoRelle, Jeffrey A.;Li, Xiaohong;Tang, Miao;Keller, Katie;Ludwig, Sara;Moresco, Eva Marie Y.;Beutler, Bruce
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