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

• Main Authors: Aisha Yesbolatova (Author), Yuichiro Saito (Author), Masato T. Kanemaki (Author)
• Format: Book
• Published: MDPI AG, 2020-05-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.3390/ph13050103; 1424-8247
• Keywords: auxin-inducible degron; conditional protein depletion; gene knockout; expression vector; Medicine; R; Pharmacy and materia medica; RS1-441
• Source: Pharmaceuticals, Vol 13, Iss 5, p 103 (2020)

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.