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Methods. 2019 Jul 15;164-165:73-80. doi: 10.1016/j.ymeth.2019.04.010. Epub 2019 Apr 24.

Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole.

Author information

1
Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka 411-8540, Japan; Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka 411-8540, Japan.
2
Department of Biochemistry, Okayama University of Science, Okayama 700-0005, Japan.
3
Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka 411-8540, Japan; Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka 411-8540, Japan. Electronic address: mkanemak@nig.ac.jp.

Abstract

Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.

KEYWORDS:

Auxin; Conditional mutant; Degradation inhibitor; Degron; Genome editing; Protein degradation

PMID:
31026591
DOI:
10.1016/j.ymeth.2019.04.010
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