Protocol for auxin-inducible depletion of the RNA- binding protein PTBP1 in mouse embryonic stem cells

被引：0
|
作者：
Kainov, Yaroslav [1 ]
Zhuravskaya, Anna [1 ]
Makeyev, Eugene, V [1 ]
机构：
[1] Kings Coll London, Ctr Dev Neurobiol, London SE1 1UL, England
来源：
STAR PROTOCOLS | 2023年 / 4卷 / 04期
基金：
英国生物技术与生命科学研究理事会;
关键词：
Cell Culture; CRISPR; Proteomics;
D O I：
10.1016/j.xpro.2023.102644
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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.1
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页数：16
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