DOI: 10.5061/dryad.g1jwstqt9
title: Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

description: 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 modeling drug
 activity in mammalian tissues.
description: <strong>Flow Cytometry</strong>
 Flow Cytometry data are provided as unprocessed .fcs files. Data
 were collected as follows. For cultured adherent cells,
 single cell suspensions were first generated using trypsin (MEFs) or
 accutase (Neural Stem Cells). For haematopoietic cells, samples were
 prepared from single-cell suspensions of bone marrow and thymus. Samples
 were incubated with fluorescently-conjugated antibodies against cell
 surface markers (Table S2) and Fixable Viability Dye (eBioscience,
 65-0865-14, 1 in 200 dilution) diluted in Flow Cytometry Staining Buffer
 (eBioscience, 00-4222-26) (20 minutes at 4 °C). Samples were then washed
 in a 10-fold volume of Flow Cytometry Staining Buffer before
 centrifugation at 300 g for 5 minutes at 4°C. Pellets were resuspended in
 Cytofix/Cytoperm solution (BD Bioscience, 554722) following manufacturer’s
 instructions and washed in Perm/Wash buffer (BD Bioscience, 554723). If
 required, samples were incubated with fluorescently conjugated antibodies
 against intracellular markers for 20 minutes at room temperature. For
 intracellular gH2AX staining, samples were further permeabilised by
 resuspending in Perm/Wash buffer (1 mL) for 15 minutes at 4 °C before
 antibody incubation. After intracellular antibody incubation, all stained
 samples were then washed in Perm/Wash buffer (300 g/ 5 minutes/ 4 °C).
 Cell Trace Yellow (Thermo Fisher C34567) experiments were conducted
 according to the manufacturer’s protocol. Samples were resuspended in DAPI
 staining solution (1 µg/mL DAPI in PBS). DAPI-stained samples were
 incubated on ice for at least 15 minutes before data
 acquisition. Data acquisition (BD LSRFortessa) was
 performed no more than 24 hours following sample fixation. Identical laser
 power was used to quantify Clover signal across all experiments. Data
 analysis was conducted using FlowJo software (Treestar). Cellular
 debris/aggregates were excluded using strict forward- and side-scatter
 gating strategies. Cell cycle stages were gated based on DNA content
 (DAPI) fluorescence. Our protein degradation experiments focused on S/G2/M
 phase cells in order to control for cell-cycle differences between cell
 types, and because condensins function primarily during cell division.
 Wild-type samples lacking Clover expression were processed and stained in
 parallel to transgenic samples. To correct for autofluorescence,
 background fluorescence was measured for each cell population from
 wild-type samples, and then subtracted from transgenic fluorescence
 values. To generate boxplots, the background-corrected fluorescence value
 from each of &gt;1000 cells was expressed relative to the mean of the
 vehicle only condition. <strong>Western
 Blots</strong> Were generated from whole cell or
 tissue protein extracts and probed with primary antibodies raised against
 proteins of interest. Image files were captured on either an Odyssey CLx
 Imaging system (LiCOR) or ImageQuant (Cytiva).
 <strong>Fluorescence Imaging</strong>
 Images of small intestine sections were acquired in 3D at 100X
 magnification using a Photometrics Coolsnap HQ2 CCD camera and a Zeiss
 AxioImager A1 fluorescence microscope with a Plan Apochromat 100x 1.4NA
 objective, a Nikon Intensilight Mercury based light source (Nikon UK Ltd,
 Kingston-on-Thames, UK) and either Chroma #89014ET (3 colour) or #89000ET
 (4 colour) single excitation and emission filters (Chroma Technology
 Corp., Rockingham, VT). Images of embryonic whole-mount
 cryosections were acquired in 2D using a Zeiss Axioscan Z1 with a
 Plan-Apochomat 40x 0.95Korr M27 objective and an Axiocam 506 camera using
 DAPI channel as focus.
description: Flow Cytometry data analysis was conducted using FlowJo software
 (Treestar). Flowing software is a free alternative: http://www.flowingsoftware.com/. 3D imaging datasets datasets were visualised and analysed for fluorescence intensity using Imaris V9.5 (Bitplane, Oxford Instruments, UK). FIJI/ImageJ is an open source alternative. 2D imaging datasets were analysed using QuPath 0.3.0, which is open source.