To investigate the role of proteins in cells, gene knock down method is widely applied. Using the Auxin Inducible Degron (AID) System, 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
Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.
Hatoyama, Y., Homma, Y., Hiragi, S., Fukuda, M.
J. Cell Sci. 134 (24): jcs259184 (2021). PMID 34817057.
| Catalog # | Name of clone | Short description |
|---|---|---|
| RDB19431 | pMRX-bsr-FLAG-OsTIR1 | Retroviral expression vector of oryza sativa TIR-L1 tagged with FLAG. |
A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.
Nishimura, K., Yamada, R., Hagihara, S., Iwasaki, R., Uchida, N., Kamura, T., Takahashi, K., Torii, K.U,. Fukagawa, T.
Nucleic Acids Res. 48 (18): e108, 2020. PMID: 32941625.
| Catalog # | Name of clone | Short description | ||
|---|---|---|---|---|
| RDB19226 | pAID1.2-CMV-NmScarlet-mAID | Expresson vector to construct mScarlet-mAID tagged target gene and OsTIR1 (WT) simultaneously by CMV promoter for the super-sensitive AID system. | ||
| RDB19227 | pAID1.2-EF1a-NmScarlet-mAID | Expresson vector to construct mScarlet-mAID tagged target gene and OsTIR1 (WT) simultaneously by EF1 alpha promoter for the super-sensitive AID system. | ||
| RDB19228 | pAIDFA-CMV-NmScarlet-mAID | Expresson vector to construct mScarlet-mAID tagged target gene and OsTIR1 mutant (F74A) simultaneously by CMV promoter for the super-sensitive AID system. | ||
| RDB19229 | pAIDFA-EF1a-NmScarlet-mAID | Expresson vector to construct mScarlet-mAID tagged target gene and OsTIR1 mutant (F74A) simultaneously by EF1 alpha promoter for the super-sensitive AID system. | ||
| RDB19230 | pAID1.2-CMV-linearizing in pX330 | Expression vector of Cas9 protein and sgRNA to linearize pAID-CMV plasmids in vivo. | ||
| RDB19231 | pAID-EF1a- linearizing in pX330 | Expression vector of Cas9 protein and sgRNA to linearize to pAID-EF1 alpha plasmids in vivo. | ||
| Catalog # | Name of clone | Short description |
|---|---|---|
| RDB16762 | pcDNA-Ostir1-pA | Expression vector of transport inhibitor response 1 derived from Oryza sativa (OsTIR). |
The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.
Yesbolatova, A., Saito, Y., Kitamoto, N., Makino-Itou, H., Ajima, R., Nakano, R., Nakaoka, H., Fukui, K., Gamo, K., Tominari, Y., Takeuchi, H., Saga, Y., Hayashi, K., Kanemaki, M.T.
Nat. Commun. 11: 5701, 2020.
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. CsTIR1 and AtAFB2 expression vectors and helper plasmid
a. CsTIR1 and AtAFB2 expression vectors
| Catalog# | Name of clone | Short description |
|---|---|---|
| RDB13918 | pMK232 | OsTIR1 expression vector, PURO |
| RDB16811 | pMK364 | OsTIR1 expression vector, loxP-PURO-loxP |
| RDB18337 | pMK381 | OsTIR1 F74G expression vector, loxP-PURO-loxP |
| RDB18347 | pMK233 | AtAFB2 expression vector, loxP-PURO-loxP |
b. Helper plasmid for knock-in a expression vector to the human AAVS1 site
| Catalog # | Name of clone | Short description |
|---|---|---|
| RDB13917 | AAVS1 T2 CRIPR in pX330 | A CRISPR/Cas plasmid for making DNA dounle-strand break at the human AAVS1 locus. |
c. CsTIR1 expression vector of yeast
| Catalog # | Name of clone | Short description |
|---|---|---|
| RDB18341 | pMK198 | GAL-OsTIR1 expression vector of yeast |
| RDB18342 | pMK419 | GAL-OsTIR1(F74G) expression vector of yeast |
| RDB18343 | pMK425 | GAL-OsTIR1(F74A) expression vector of yeast |
Table 2. Tags
a. mAID tags, 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. mAID tags, 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 3. Tags
a. Tags without mAID, N-terminal tagging
| Selection marker |
Attached tag at the N-terminus | ||
|---|---|---|---|
| mClover | mCherry2 | S tag-3FLAG | |
| Hygro | pMK376 | pMK377 | pMK375 |
b. Tags without mAID, 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 4. mAID and mIAA7 tagging specific gene
| Gene | Helper | Tagging with Hygro marker | Tagging with Neo marker |
|---|---|---|---|
| BRD4 | BRD4-N CRISPR | mAID-BRD4 donor | |
| CTCF | CTCF-C CRISPR | CTCF-mAC donor (Hygro) | CTCF-mAC donor (Neo) |
| DHC1 | DHC1 CRISPR | pMK297 | pMK296 |
| DHC1 | DHC1 CRISPR | DHC1-mIAA7 Hygro | DHC1-mIAA7 Neo |
| RAD21 | RAD21 C-tag CIRPSR | pMK265 | pMK262 |
| SMC2 | SMC2 CRISPR | SMC2-mAC donor (Hygro) | |
| TOP2A | pMK312 | pMK322 | pMK321 |
Table 5. Simultaneous expression of OsTIR1 and mAID-EGFP
| Catalog no. | Name of clone | Short description |
|---|---|---|
| RDB18338 | pAAV-hSyn-OsTIR1(WT) | Adeno-associated virus (AAV) expression vector of OsTIR1(wild type) and mAID-EGFP-NES marker with hSyn promoter. |
| RDB18339 | pAAV-hSyn-OsTIR1(F74G) | Adeno-associated virus (AAV) expression vector of OsTIR1(F74G) and mAID-EGFP-NES marker with hSyn promoter. |
| RDB18340 | pAAV-hSyn-OsTIR1(F74A) | Adeno-associated virus (AAV) expression vector of OsTIR1(F74A) and mAID-EGFP-NES marker with hSyn promoter. |
| RDB18333 | pAY7 | TOL2 expression vector of OsTIR1(WT) and mAID-EGFP-NLS marker. |
| RDB18334 | pAY15 | TOL2 expression vector of OsTIR1(F74G) and mAID-EGFP-NLS marker. |
| RDB18335 | pAY19 | TOL2 expression vector of OsTIR1(F74A) and mAID-EGFP-NLS marker. |
| RDB18363 | pMK427 | TOL2 expression vector of OsTIR1(WT) and mAID-EGFP-Nluc marker. |
| RDB18364 | pMK411 | TOL2 expression vector of OsTIR1(F74G) and mAID-EGFP-Nluc marker. |
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)
References & Related Articles
- Nishimura, K., Yamada, R., Hagihara, S., Iwasaki, R., Uchida, N., Kamura, T., Takahashi, K., Torii, K.U,. Fukagawa, T. A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair. Nucleic Acids Res. 48 (18): e108, 2020. PMID: 32941625.
- 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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2023.04.06
