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Clontech Laboratories, Inc. and RIKEN BioResource Research Center concluded license agreement on preservation and distribution of Fluorescent Proteins, DsRed2 and mCherry for academic use

BRC News

Kanemaki’s Auxin Inducible Degron (AID) System


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)

map of RDB08468

 


 

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References & Related Articles

  1. 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.
  2. Research highlights
    Development of a CRISPR/Cas9-based tagging method and a degradation inhibitor for the auxin-inducible degron (AID) system
  3. 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.
  4. 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.
  5. 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.
  6. 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
  7. 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
  8. Kanemaki, M.T. Frontiers of protein expression control with conditional degrons. Eur. J. Physiol., 465, 419-425, 2013. PMID: 23271452.
  9. 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
  10. 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.
  11. Matato Kanemaki Lab Web Page
  12. BioResource Now! Issue 7, July 2011 (PDF, in Japanese)
  13. BioResource Now! Issue 7, July 2011 (PDF, in English)

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(GRP0016e 2012.11.07 T.M.)

2019.05.16