Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK.
Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK.
STAR Protoc. 2023 Dec 15;4(4):102644. doi: 10.1016/j.xpro.2023.102644. Epub 2023 Oct 19.
Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets. For complete details on the use and execution of this protocol, please refer to Iannone et al..
诱导靶蛋白降解为功能研究提供了一种强大的方法。本文提供了一种在小鼠胚胎干细胞（ESCs）中严格控制 RNA 结合蛋白 PTBP1 耗竭的方案。我们描述了建立表达诱导型色氨酸受体蛋白 OsTIR1 和利用 CRISPR-Cas9 和同源定向修复试剂对内源性 Ptbp1 等位基因进行标记的 ESCs 系的步骤。然后，我们详细介绍了通过免疫印迹检测诱导性 PTBP1 敲低效率的程序。该方案适用于其他蛋白质靶标。有关此方案使用和执行的完整详细信息，请参阅 Iannone 等人的研究。
