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pNHK60 (#RDB08468)

Expression clone for an auxin-based degron (AID) system, GFP-aid-NLS.

Alternative name OsTIR1-9Myc::IRES::GFP-aid-NLS (kan/neoR)
Clone info. Expression clone for an auxin-based degron (AID) system. GFP-aid-NLS. Refer Supplementary figure 10 (pIRES OsTIR1-EGFP-IAA17) of Nat. Methods, 6, 917-922, 2009; PMID: 19915560.
Comment Please note that (1) the degron system may not work well in T-antigen expressing cells such as HEK293T because pNHK60 has the SV40 replication origin and the expression level of GFP-degron is quite high, (2) the depositor found that degradation is efficient in stable cells rather than in transiently transiently transfected cells. Accession numbers, OsTIR1 (GeneID 4335696); AtIAA17 (GeneID 839568). To make 0.5 M stock solution, dissolve indole-3-acetic acid (IAA) (Sigma-Aldrich, 45533) or 1-naphthaleneacetic acid (NAA) (Sigma-Aldrich, 35745) in 100 % DMSO. IAA solution can be made in 100 % ethanol. Aliquots can be kept for 6 months in the dark at -20oC (Devrekanli, A., Kanemaki, M.T. Methods in Molecular Biology, 1369, 257-278, 2016).
Vector backbone pIRES-EGFP (Plasmid)
Selectable markers Kan^r
Growth conditions 37C, LB+Kan
Gene/insert name Oryza sativa OsTIR1 cDNA Arabidopsis thaliana AtIAA17 cDNA Aequorea victoria GFP cDNA
Depositor|Developer Kanemaki, Masato |
 
Sequence (full) RDB08468hts01.seq checkAssembled from experimentally sequenced data.
Remarks, protocol and/or map (pdf) RDB08468.pdf
Publication Nishimura, K., Nat Methods 6(12), 917-922, (2009). PMID 19915560. [link to RRC of NBRP]
Publication Kanemaki, M., Patent Abstracts WO2010125620A1. [link to RRC of NBRP]
Test sheet RDB08468_A5Lbp1-2.pdf 
 

Distribution information

Please check terms and conditions set forth by the depositor, which are specified in the RIKEN BRC Catalog and/or Web Catalog.
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Please visit Information of Request for Distribution.[link] 
Terms and conditions for distribution In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the following literature(s) designated by the DEPOSITOR is requested (Nishimura, K., Nat. Methods., 6, 917-922 (2009)). This DNA clone is used for academic research purpose only. This is not to be used on a commercial basis. For use of this DNA clone except for internal academic research, the RECIPIENT must contact a NIG license representative at chizai@nig.ac.jp.
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必要書類
提供依頼書  [依頼書の記入例 ]
提供同意書 (MTA, 非営利学術目的用)[Word] [MTAの記入例 ]
提供同意書 (MTA, 営利目的用)[Word]
手続きの概要は、「提供申込みについて[link]」をご覧ください。
MTAに書く使用条件 利用者は、研究成果の公表にあたって寄託者の指定する文献を引用する (Nishimura, K., Nat. Methods., 6, 917-922 (2009))。学術研究にのみ限定、商業営利目的利用を禁ずる。学術内部利用以外の利用の際は、国立遺伝学研究所 (chizai@nig.ac.jp) に相談すること。

Catalog # Resource name Shipping form Fee (non-profit org.)
RDB08468 pNHK60 DNA solution JPY 9,460 (not-for-profit academic purpose)
JPY 18,920 (for-profit-research purpose)
plus cost of shipping containers, dry ice (if required) and shipping charge

How to cite this biological resource

Materials & Methods section:

The pNHK60 was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan (cat. RDB08468).

Reference section:

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(12), 917-922, (2009). PMID 19915560. [link to RRC of NBRP]
Kanemaki, M., Kakimoto, T., Nishimura, K., Takisawa, H., Fukagawa, T., Method for inducing degradation of protein in mammalian cell. Patent Abstracts WO2010125620A1. [link to RRC of NBRP]

Further references such as user reports and related articles (go to bottom)

References

Original, user report and related articles

original Nishimura, K., An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12), 917-922, (2009). PMID 19915560. [link to RRC of NBRP]
original Kanemaki, M., Method for inducing degradation of protein in mammalian cell. Patent Abstracts WO2010125620A1. [link to RRC of NBRP]
reference Devrekanli, A., Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes. Methods in Molecular Biology 1369: 257-278 (2016). PMID 26519318. [link to Journal]
reference Delacour, Q., Light-Activated Proteolysis for the Spatiotemporal Control of Proteins. ACS Chem. Biol.10 (7): 1643-1647 (2015). PMID 25938742. [link to Journal]
reference Wend, S., A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics. Sci. Rep. 3: 2052 (2014). PMID 23787479. [link to Journal]
reference Farr, C.J., The alpha isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells. Nucleic Acids Res. 42 (7): 4414-4426 (2014). PMID 24476913. [link to Journal]
reference Nishimura, K., Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin-Inducible Degron (AID). Curr. Protoc. Cell Biol. \64: 20.9.1-16 (2014). PMID 25181302. [link to Journal]
reference Kampf, M.M., Rewiring and dosing of systems modules as a design approach for synthetic mammalian signaling networks. Mol. Biosyst. 8 (6): 1824-1832 (2012). PMID 22532387. [link to Journal]
reference Holland, A.J., Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 109 (49): E3350-7 PMID 23150568. [link to Journal]