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

Citation

Wood, Andrew (2022), Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons, Dryad, Dataset, https://doi.org/10.5061/dryad.g1jwstqt9

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 modeling drug activity in mammalian tissues.

Methods

Flow Cytometry

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 >1000 cells was expressed relative to the mean of the vehicle only condition.

Western Blots

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).

Fluorescence Imaging

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.

Usage Notes

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.

Funding

Medical Research Council

Wellcome Trust

Canadian Institutes of Health Research