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

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. Howe...

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Bibliographic Details
Published in:Pharmaceuticals (Basel, Switzerland) Switzerland), 2020-05, Vol.13 (5), p.103
Main Authors: Yesbolatova, Aisha, Saito, Yuichiro, Kanemaki, Masato T
Format: Article
Language:English
Subjects:
auxin-inducible degron
Cloning
conditional protein depletion
CRISPR
Deoxyribonucleic acid
DNA
expression vector
gene knockout
Genomes
Plasmids
Proteins
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Summary: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.
ISSN:1424-8247
1424-8247
DOI:10.3390/ph13050103