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オーキシンデグロン法 (AID system) によるタンパク質発現制御

  • 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)

construction images

参考文献・関連文献

  1. 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.
  2. 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.
  3. 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
  4. 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.
  5. 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.
  6. 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～
  7. 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.
  8. 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
  9. 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
  10. Kanemaki, M.T. Frontiers of protein expression control with conditional degrons. Eur. J. Physiol., 465, 419-425, 2013. PMID: 23271452.
  11. 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
  12. 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.
  13. Masato Kanemaki Lab Resources
  14. 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]
  15. BioResource Now! Issue 7, July 2011 (PDF, in Japanese)
  16. BioResource Now! Issue 7, July 2011 (PDF, in English)

(GRP0016j 2014.11.11 T.M.)

2023.04.06



 

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