Reference - Detail
| RRC ID | 55760 |
|---|---|
| Author | Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT. |
| Title | Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. |
| Journal | Methods |
| 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. |
| Volume | 164-165 |
| Pages | 73-80 |
| Published | 2019-7-15 |
| DOI | 10.1016/j.ymeth.2019.04.010 |
| PII | S1046-2023(18)30331-1 |
| PMID | 31026591 |
| MeSH | CRISPR-Cas Systems / genetics Cell Culture Techniques / methods Genetic Vectors / genetics HCT116 Cells Half-Life Humans Indoleacetic Acids / pharmacology* Luminescent Proteins / genetics Luminescent Proteins / metabolism Plant Proteins / antagonists & inhibitors Plant Proteins / genetics Plant Proteins / metabolism Plasmids / genetics Proteolysis / drug effects* Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism* Transfection / methods Ubiquitin-Protein Ligases / antagonists & inhibitors Ubiquitin-Protein Ligases / genetics Ubiquitin-Protein Ligases / metabolism |
| IF | 3.782 |
| Times Cited | 7 |
| Resource | |
| DNA material | pMK344 (Hygro-P2A-mAID) (RDB16805) pMK345 (Hygro-P2A-mAID-mClover) (RDB16806) pMK346 (Hygro-P2A-mAID-mCherry) (RDB16807) pMK347 (BSD-P2A-mAID) (RDB16808) pMK348 (BSD-P2A-mAID-mClover) (RDB16809) pMK349 (BSD-P2A-mAID-mCherry) (RDB16810) pMK364 (CMV-OsTIR1-loxP-PURO-loxP) (RDB16811) pMK375 (Hygro-P2A-Stag-3FLAG) (RDB16813) pMK376 (Hygro-P2A-mClover) (RDB16814) pMK377 (Hygro-P2A-mCherry2) (RDB16815) pMK388 (mClover-BSD) (RDB16816) pMK389 (mCherry-BSD) (RDB16817) pMK390 (Stag-3FLAG-BSD) (RDB16818) pMK391 (mAID-BSD) (RDB16819) pMK392 (mAID-mClover-BSD) (RDB16820) pMK393 (mAID-mCherry-BSD) (RDB16821) pMK394 (mAID-Nluc-Neo) (RDB16822) pMK401 (Hygro-P2A-mAID-Nluc) (RDB16823) pMK407 (BSD-P2A-mAID-Nluc) (RDB16824) pMK408 (mAID-Nluc Hygro) (RDB16825) pMK409 (mAID-Nluc BSD) (RDB16826) |