Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Lewis MacDonald; Gillian Taylor; Jennifer Brisbane; Ersi Christodoulou; Lucy Scott; Alexander von Kriegsheim; Janet Rossant; Bin Gu; Andrew J Wood

doi:10.7554/eLife.77987

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Lewis MacDonald, Gillian Taylor, Jennifer Brisbane, Ersi Christodoulou, Lucy Scott, Alexander von Kriegsheim, Janet Rossant, Bin Gu, Andrew J Wood^*

^*Corresponding author for this work

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

Abstract / Description of output

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

Original language	English
Journal	eLIFE
Volume	11
DOIs	https://doi.org/10.7554/eLife.77987
Publication status	Published - 23 Jun 2022

Keywords / Materials (for Non-textual outputs)

Animals
Chromosomes/metabolism
Indoleacetic Acids/metabolism
Ligands
Mammals/metabolism
Mice
Proteolysis
Ubiquitin-Protein Ligases/metabolism

Access to Document

10.7554/eLife.77987

elife-77987-v2
Copyright Macdonald, Taylor et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.DOI: https://doi.org/10.7554/eLife.77987
Final published version, 1.86 MB

1 Finished

Chromosomal instability during normal and condensin II deficient haematopoiesis
Wood, A.
UK-based charities
1/02/14 → 31/01/19
Project: Research

1 Preprint

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
Wood, A. J., MacDonald, L. & Taylor, G., 13 Jan 2022, bioRxiv, at Cold Spring Harbor Laboratory.
Research output: Working paper › Preprint

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
Wood, A. (Creator), Dryad, 2022
DOI: 10.5061/dryad.g1jwstqt9, http://datadryad.org/stash/dataset/doi:10.5061/dryad.g1jwstqt9
Dataset

Bioresearch and Veterinary Services (BVS) Central Transgenic Core
Martha Koerner (Manager)
Facility/equipment: Facility
Institute for Genetics and Cancer Mass Spectrometry Facility
Alex Von Kriegsheim (Manager)
Deanery of Molecular, Genetic and Population Health Sciences
Facility/equipment: Facility

Cite this

@article{1ec5d6852a68462481837253cfac4b29,

title = "Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons",

abstract = "Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.",

keywords = "Animals, Chromosomes/metabolism, Indoleacetic Acids/metabolism, Ligands, Mammals/metabolism, Mice, Proteolysis, Ubiquitin-Protein Ligases/metabolism",

author = "Lewis MacDonald and Gillian Taylor and Jennifer Brisbane and Ersi Christodoulou and Lucy Scott and {von Kriegsheim}, Alexander and Janet Rossant and Bin Gu and Wood, {Andrew J}",

note = "Funding Information: We thank the University of Edinburgh BVS Central Transgenic Core for performing CRISPR microinjections to generate AID:Clover-tagged mouse lines, the Biological Research Facility at the Western General Hospital for animal husbandry, the IGC Flow Cytometry Facility, IGC Advanced Imaging Resource and IGC Mass Spectrometry facility for technical support. We acknowledge technical support from the Model Production Core staff led by M Gertsenstein at the Centre for Phenogenomics for generating the TIR1 knock-in mouse lines and Eszter Posfai (Princeton University) for early collaboration efforts on proof-of-principle studies on AID in mouse embryos. We are grateful to Luke Boulter for antibodies and advice on whole-mount immunofluorescence, to Wendy Bickmore for comments on the manuscript and to Gopal Sapkota for useful discussions. We also acknowledge Masato Kane-maki and co-workers for their pioneering efforts to develop the auxin-inducible degron system. This work was supported by a Sir Henry Dale Fellowship from the Wellcome Trust (AW 102560/Z/13/Z), an MRC Unit award to the MRC Human Genetics Unit, and Funding from CIHR (JR FDN-143334). Funding Information: Funder Grant reference number Author Medical Research Council Unit award to the MRC Human Genetics Unit Lewis Macdonald Gillian C Taylor Jennifer Margaret Brisbane Ersi Christodoulou Lucy Scott Andrew J Wood Medical Research Council MC_PC_21040 Andrew J Wood Gillian C Taylor Wellcome Trust 102560/Z/13/Z Lewis Macdonald Gillian C Taylor Jennifer Margaret Brisbane Ersi Christodoulou Lucy Scott Andrew J Wood Canadian Institutes of Health Research JR FDN-143334 Janet Rossant Bin Gu The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Publisher Copyright: ‍@ Macdonald, Taylor.",

year = "2022",

month = jun,

day = "23",

doi = "10.7554/eLife.77987",

language = "English",

volume = "11",

journal = "eLIFE",

issn = "2050-084X",

publisher = "ELIFE SCIENCES PUBLICATIONS LTD",

}

TY - JOUR

T1 - Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

AU - MacDonald, Lewis

AU - Taylor, Gillian

AU - Brisbane, Jennifer

AU - Christodoulou, Ersi

AU - Scott, Lucy

AU - von Kriegsheim, Alexander

AU - Rossant, Janet

AU - Gu, Bin

AU - Wood, Andrew J

N1 - Funding Information: We thank the University of Edinburgh BVS Central Transgenic Core for performing CRISPR microinjections to generate AID:Clover-tagged mouse lines, the Biological Research Facility at the Western General Hospital for animal husbandry, the IGC Flow Cytometry Facility, IGC Advanced Imaging Resource and IGC Mass Spectrometry facility for technical support. We acknowledge technical support from the Model Production Core staff led by M Gertsenstein at the Centre for Phenogenomics for generating the TIR1 knock-in mouse lines and Eszter Posfai (Princeton University) for early collaboration efforts on proof-of-principle studies on AID in mouse embryos. We are grateful to Luke Boulter for antibodies and advice on whole-mount immunofluorescence, to Wendy Bickmore for comments on the manuscript and to Gopal Sapkota for useful discussions. We also acknowledge Masato Kane-maki and co-workers for their pioneering efforts to develop the auxin-inducible degron system. This work was supported by a Sir Henry Dale Fellowship from the Wellcome Trust (AW 102560/Z/13/Z), an MRC Unit award to the MRC Human Genetics Unit, and Funding from CIHR (JR FDN-143334). Funding Information: Funder Grant reference number Author Medical Research Council Unit award to the MRC Human Genetics Unit Lewis Macdonald Gillian C Taylor Jennifer Margaret Brisbane Ersi Christodoulou Lucy Scott Andrew J Wood Medical Research Council MC_PC_21040 Andrew J Wood Gillian C Taylor Wellcome Trust 102560/Z/13/Z Lewis Macdonald Gillian C Taylor Jennifer Margaret Brisbane Ersi Christodoulou Lucy Scott Andrew J Wood Canadian Institutes of Health Research JR FDN-143334 Janet Rossant Bin Gu The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Publisher Copyright: ‍@ Macdonald, Taylor.

PY - 2022/6/23

Y1 - 2022/6/23

N2 - Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

AB - Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

KW - Animals

KW - Chromosomes/metabolism

KW - Indoleacetic Acids/metabolism

KW - Ligands

KW - Mammals/metabolism

KW - Mice

KW - Proteolysis

KW - Ubiquitin-Protein Ligases/metabolism

U2 - 10.7554/eLife.77987

DO - 10.7554/eLife.77987

M3 - Article

C2 - 35736539

SN - 2050-084X

VL - 11

JO - eLIFE

JF - eLIFE

ER -

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

Access to Document

Fingerprint

Projects

Chromosomal instability during normal and condensin II deficient haematopoiesis

Research output