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

  1. Lewis Macdonald
  2. Gillian C Taylor
  3. Jennifer Margaret Brisbane
  4. Ersi Christodoulou
  5. Lucy Scott
  6. Alex von Kriegsheim
  7. Janet Rossant
  8. Bin Gu  Is a corresponding author
  9. Andrew J Wood  Is a corresponding author
  1. University of Edinburgh, United Kingdom
  2. Hospital for Sick Children, Canada
  3. Michigan State University, United States

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 condensin 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.

Data availability

Proteomics data underlying Figure 6B have been submitted to the PRIDE database under accession PXD032374. All other primary data, including flow cytometry files, fluorescence imaging and uncropped western blot scans are available through the DRYAD digital repository at https://doi.org/10.5061/dryad.g1jwstqt9. Requests for the Rosa26Tir1 transgenic mouse line should be addressed to Bin Gu (guibin1@msu.edu), and requests for the Ncaph- and Ncaph2-AID:Clover lines should be addressed to Andrew Wood (Andrew.j.wood@ed.ac.uk).

The following data sets were generated

Article and author information

Author details

  1. Lewis Macdonald

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Gillian C Taylor

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Jennifer Margaret Brisbane

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4943-5331
  4. Ersi Christodoulou

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Lucy Scott

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Alex von Kriegsheim

    Cancer Research UK Edinburgh Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4952-8573
  7. Janet Rossant

    Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3731-5466
  8. Bin Gu

    Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Lansing, United States
    For correspondence
    gubin1@msu.edu
    Competing interests
    The authors declare that no competing interests exist.
  9. Andrew J Wood

    MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    For correspondence
    andrew.wood@igmm.ed.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0653-2070

Funding

Medical Research Council

  • Lewis Macdonald
  • Gillian C Taylor
  • Jennifer Margaret Brisbane
  • Ersi Christodoulou
  • Lucy Scott
  • Andrew J Wood

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

Medical Research Council (MC_PC_21040)

  • Andrew J Wood

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal work was approved by a University of Edinburgh internal ethics committee and was performed in accordance with institutional guidelines under license by the UK Home Office. AID knock-in alleles were generated under project license PPL 60/4424. Rosa26Tir1 knockin mouse lines were generated under the Canadian Council on Animal Care Guidelines for Use of Animals in Research and Laboratory Animal Care under protocols approved by the Centre for Phenogenomics Animal Care Committee (20-0026H). Experiments involving double transgenic animals were conducted under the authority of UK project license PPL P16EFF7EE

Reviewing Editor

  1. Guillaume Pavlovic, PHENOMIN, Institut Clinique de la Souris (ICS), CELPHEDIA, France

Publication history

  1. Received: February 18, 2022
  2. Accepted: May 19, 2022
  3. Accepted Manuscript published: June 23, 2022 (version 1)

Copyright

© 2022, Macdonald et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Lewis Macdonald
  2. Gillian C Taylor
  3. Jennifer Margaret Brisbane
  4. Ersi Christodoulou
  5. Lucy Scott
  6. Alex von Kriegsheim
  7. Janet Rossant
  8. Bin Gu
  9. Andrew J Wood
(2022)
Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
eLife 11:e77987.
https://doi.org/10.7554/eLife.77987

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