Academic Journal
Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
| Title: | Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector |
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| Authors: | Aisha Yesbolatova, Yuichiro Saito, Masato T. Kanemaki |
| Source: | Pharmaceuticals, Vol 13, Iss 5, p 103 (2020) |
| Publisher Information: | MDPI AG, 2020. |
| Publication Year: | 2020 |
| Collection: | LCC:Medicine LCC:Pharmacy and materia medica |
| Subject Terms: | auxin-inducible degron, conditional protein depletion, gene knockout, expression vector, Medicine, Pharmacy and materia medica, RS1-441 |
| More Details: | 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. |
| Document Type: | article |
| File Description: | electronic resource |
| Language: | English |
| ISSN: | 1424-8247 |
| Relation: | https://www.mdpi.com/1424-8247/13/5/103; https://doaj.org/toc/1424-8247 |
| DOI: | 10.3390/ph13050103 |
| Access URL: | https://doaj.org/article/65718b3a72cb434d9ff7e7aa6a41d21a |
| Accession Number: | edsdoj.65718b3a72cb434d9ff7e7aa6a41d21a |
| Database: | Directory of Open Access Journals |
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| ISSN: | 14248247 |
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| DOI: | 10.3390/ph13050103 |
| Published in: | Pharmaceuticals |
| Language: | English |