To investigate the role of proteins in cells, gene knock down method is widely applied. Here Dr. Kanemaki offers a new method of reduction of interested proteins. He provides a novel method Auxin Inducible Degron (AID) System with which you can degrade proteins fused with a degron (a small domain that can induce protein degradation) in a very short period of time.
DNA Resource
Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole.
Yesbolatova, A., Natsume, T., Hayashi, K.I., Kanemaki, M.T.
Methods pii: S1046-2023 (18) 30331-1, 2019. PMID: 31026591.
Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.
Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T.
Cell Reports 15: 210-218, 2016. PMID: 27052166.
Table 1. mAID tags
a. N-terminal tagging.
| Selection marker |
Attached tag at the N-terminus | |||
|---|---|---|---|---|
| mAID | mAID-mClover | mAID-mCherry2 | mAID-Nluc | |
| Hygro | pMK344 | pMK345 | pMK346 | pMK401 |
| BSD | pMK347 | pMK348 | pMK349 | pMK407 |
b. C-terminal tagging.
| Selection marker |
Attached tag at the C-terminus | |||
|---|---|---|---|---|
| mAID | mAID-mClover | mAID-mCherry2 | mAID-Nluc | |
| Neo | pMK286 | pMK289 | pMK292 | pMK394 |
| Hygro | pMK287 | pMK290 | pMK293 | pMK408 |
| BSD | pMK391 | pMK392 | pMK393 | pMK409 |
| Bsr | pMK288 | pMK291 | pMK294 | |
Table 2. Tags without mAID.
a. N-terminal tagging.
| Selection marker |
Attached tag at the N-terminus | ||
|---|---|---|---|
| mClover | mCherry2 | S tag-3FLAG | |
| Hygro | pMK376 | pMK377 | pMK375 |
b. C-terminal tagging.
| Selection marker |
Attached tag at the C-terminus | ||
|---|---|---|---|
| mClover | mCherry2 | S tag-3FLAG | |
| Neo | pMK277 | pMK280 | pMK283 |
| Hygro | pMK278 | pMK281 | pMK284 |
| BSD | pMK388 | pMK389 | pMK390 |
| Bsr | pMK279 | pMK282 | pMK285 |
Table 3. CsTIR1 expression vectors.
| Catalog# | Name of clone | Running title |
|---|---|---|
| RDB13918 | pMK232 | OsTIR1 expression vector, Puro |
| RDB13919 | pMK243 | OsTIR1 expression vector, Puro |
| RDB16811 | pMK364 | OsTIR1 expression vector, loxP-Puro-loxP |
| RDB16812 | pMK365 | OsTIR1 expression vector, loxP-Puro-loxP |
Table 4. Helper plasmid for knock-in a expression vector to the human AAVS1 site.
| RDB13917 | AAVS1 T2 CRIPR in pX330 | A CRISPR/Cas plasmid for making DNA dounle-strand break at the human AAVS1 locus. |
An auxin-based degron system for the rapid depletion of proteins in nonplant cells.
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M.
Nat. Methods., 6, 917-922, 2009. PMID: 19915560.
| Catalog# | Name of clone | Running title | Type |
|---|---|---|---|
| RDB08468 | pNHK60 | Expression clone for an auxin-based degron (AID) system | Plasmid |
| RDB08469 | pMK106 | Expression clone for an auxin-based degron (AID) system | Plasmid |
| RDB08470 | pMK107 | Expression clone for an auxin-based degron (AID) system | Plasmid |
Please refer
Supplementary Figure 11 for pMK106 (aid-CENPH)
Supplementary Figure 11 for pMK107 (CENPH-aid)
Supplementary figure 10 for pNHK60 (pIRES OsTIR1-EGFP-IAA17)
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References & Related Articles
- Yesbolatova, A., Natsume, T., Hayashi, K.I., Kanemaki, M.T. Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. Methods pii: S1046-2023 (18) 30331-1, 2019. PMID: 31026591.
- Research highlights
Development of a CRISPR/Cas9-based tagging method and a degradation inhibitor for the auxin-inducible degron (AID) system - Natsume, T., Nishimura, K., Minocherhomji, S., Bhowmick, R., Hickson, I.D., Kanemaki, M.T. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis. Genes Dev. 31 (8): 816-829, 2017.. PMID: 28487407.
- Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Reports 15: 210-218, 2016. PMID: 27052166.
- Takagi, M., Natsume, T., Kanemaki, M.T., Imamoto, N. Perichromosomal protein Ki67 supports mitotic chromosome architecture. Genes Cells 21 (10): 1113-1124, 2016. PMID: 27610954.
- Samejima, K., Ogawa, H., Ageichik, A.V., Peterson, K.L., Kaufmann, S.H., Kanemaki, M.T., Earnshaw, W.C. Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation, Caspase Activation, and Cell Death: A CELL SUICIDE MODULE. J. Biol. Chem. 289 (45): 31617-31623, 2014. PMID: 25248749
- Rodriguez-Bravo, V., Maciejowski, J., Corona, J., Buch, H.K., Collin, P., Kanemaki, M.T., Shah, J.V., Jallepalli, P.V. Nuclear pores protect genome integrity by assembling a premitotic and Mad1-dependent anaphase inhibitor. Cell 156 (5): 1017-1031, 2014. PMID: 24581499
- Kanemaki, M.T. Frontiers of protein expression control with conditional degrons. Eur. J. Physiol., 465, 419-425, 2013. PMID: 23271452.
- Holland, A.J., Fachinetti, D., Han, J.S., Cleveland, D.W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 109 (49): E3350-E3357, 2012. PMID: 23150568
- Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods., 6, 917-922, 2009. PMID: 19915560.
- Matato Kanemaki Lab Web Page
- BioResource Now! Issue 7, July 2011 (PDF, in Japanese)
- BioResource Now! Issue 7, July 2011 (PDF, in English)
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2019.05.16