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

Yaroslav Kainov; Anna Zhuravskaya; Eugene Makeyev

doi:10.1016/j.xpro.2023.102644

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

Yaroslav Kainov, Anna Zhuravskaya, Eugene Makeyev

Developmental Neurobiology

Research output: Contribution to journal › Article › peer-review

Abstract

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

Original language	English
Article number	102644
Journal	STAR Protocols
Volume	4
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2023.102644
Publication status	Published - 15 Dec 2023

Keywords

Cell Culture
CRISPR
Proteomics

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10.1016/j.xpro.2023.102644Licence: CC BY

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abstract = "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",

keywords = "Cell Culture, CRISPR, Proteomics",

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AU - Kainov, Yaroslav

AU - Zhuravskaya, Anna

AU - Makeyev, Eugene

N1 - Funding Information: We thank Masato Kanemaki, Michael Kyba, and Feng Zhang for reagents. This work was supported by the Biotechnology and Biological Sciences Research Council ( BB/M001199/1 , BB/M007103/1 , and BB/V006258/1 ). Publisher Copyright: © 2023 The Author(s)

PY - 2023/12/15

Y1 - 2023/12/15

N2 - 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

AB - 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

KW - Cell Culture

KW - CRISPR

KW - Proteomics

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

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