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Open AccessArticle

Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector

1
Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka 411-8540, Japan
2
Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka 411-8540, Japan
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Pharmaceuticals 2020, 13(5), 103; https://doi.org/10.3390/ph13050103
Received: 17 April 2020 / Revised: 22 May 2020 / Accepted: 22 May 2020 / Published: 23 May 2020
(This article belongs to the Special Issue Targeted Protein Degradation: From Chemical Biology to Drug Discovery)
Conditional degron-based methods are powerful for studying protein function because a degron-fused protein can be rapidly and efficiently depleted by adding a defined ligand. Auxin-inducible degron (AID) is a popular technology by which a degron-fused protein can be degraded by adding an auxin. However, compared with other technologies such as dTAG and HaloPROTAC, AID is complicated because of its two protein components: OsTIR1 and mAID (degron). To simplify the use of AID in mammalian cells, we constructed bicistronic all-in-one plasmids that express OsTIR1 and a mAID-fused protein using a P2A self-cleavage sequence. To generate a HeLa mutant line for the essential replication factor MCM10, we transfected a CRISPR-knockout plasmid together with a bicistronic plasmid containing mAID-fused MCM10 cDNA. After drug selection and colony isolation, we successfully isolated HeLa mutant lines, in which mAID–MCM10 was depleted by the addition of indole-3-acetic acid, a natural auxin. The bicistronic all-in-one plasmids described in this report are useful for controlling degradation of a transgene-derived protein fused with mAID. These plasmids can be used for the construction of conditional mutants by combining them with a CRISPR-based gene knockout. View Full-Text
Keywords: auxin-inducible degron; conditional protein depletion; gene knockout; expression vector auxin-inducible degron; conditional protein depletion; gene knockout; expression vector
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Yesbolatova, A.; Saito, Y.; Kanemaki, M.T. Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector. Pharmaceuticals 2020, 13, 103.

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