- オーキシン依存的に目的タンパク質を分解することで、発現制御します。
- 培養細胞にシロイヌナズナ由来aidタグを付加した目的タンパク質とイネF-boxタンパク質TIR1をあらかじめ同時に発現させておき、培地にオーキシンを添加することで発現タンパク質の分解を誘導します。
クローン一覧
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. |
Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.
Miura, K., Matoba, S., Ogonuki, N., Namiki, T., Ito, J., Kashiwazaki, N., Ogura, A.
J. Reprod. Dev. 64 (4): 319-326 (2018). PMID 29731504.
| 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 no. | Name of clone | Short description | 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 |
- Supplementary Figure 11 for pMK106 (aid-CENPH)
- Supplementary Figure 11 for pMK107 (CENPH-aid)
- Supplementary figure 10 for pNHK60 (pIRES OsTIR1-EGFP-IAA17)
参考文献・関連文献
- 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
オーキシンデグロン(AID)法のためのCRISPR/Cas9を利用した新規タグ導入と分解阻害剤の開発 - 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.
- プレスリリース
壊れた「DNAのファスナー」を修理するには？～細胞がDNAをコピーする際のバックアップシステムを発見～ - 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.
- Masato Kanemaki Lab Resources
- プレスリリース
取り除けば働きがわかる！～特定のヒト細胞内タンパク質を素早く取り除いて機能を探る方法を開発～
プレスリリース資料 [PDF] - BioResource Now! Issue 7, July 2011 (PDF, in Japanese)
- BioResource Now! Issue 7, July 2011 (PDF, in English)
(GRP0016j 2014.11.11 T.M.)
2023.04.06
