RDB14601 - pMRX-IP-GFP-LC3-RFP

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pMRX-IP-GFP-LC3-RFP (#RDB14601)

Fluorescent probe to evaluate autophagic flux by the rat LC3 fused with GFP and RFP.

Clone info.	Fluorescent probe to evaluate autophagic flux. Expression vector of rat microtubule-associated protein 1 light chain 3 beta (LC3) fused with GFP and RFP. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP. The GFP-LC3 is degraded by autophagy, while RFP remains in the cytosol as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Please refer DISCUSSION section of Kaizuka, T., Mol. Cell 64, 835-849, 2016 for the difference between pMRX-IP-GFP-LC3-RFP-LC3deltaG and this clone.
Comment	Expression was confirmed by the depositor with immunoblotting and fluorescent microscopy. Please refer Saitoh, T. et al., FEBS Lett., 532 (1-2): 45-51, (2002) for the construction of pMRX-IP vector.
Vector backbone	pMRX-IP (Plasmid)
Selectable markers	Amp^r (E. coli), Puro^r (mammalian cells).
Growth conditions	LB+Amp, 37oC
Gene/insert name	Rat LC3 cDNA Aequorea victoria GFP cDNA Discosoma sp. RFP cDNA
Depositor\|Developer	Mizushima, Noboru \|

Sequence (full)	RDB14601hts01.seq Assembled from experimentally sequenced data.
Publication	Morishita, H., Autophagy 13 (4): 757-758 (2017). PMID 28121224. [link to RRC of NBRP]
Publication	Kaizuka, T., Mol Cell. 2016 Nov 17;64(4):835-849 (2016). PMID 27818143. [link to RRC of NBRP]
Test sheet	RDB19179_B1Gcp1-2.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.

Ordering forms
Order form [Credit Card Payment] [Bank Transfer Payment] [Example of order form ]
MTA, for use for not-for-profit academic purpose [Word] [Example of MTA ]
MTA, for use for for-profit purpose [Word]
Approval form by depositor [Word]
Please visit Information of Request for Distribution.[link]

Terms and conditions for distribution (1) BIOLOGICAL RESOURCE is limited to for academic research in non-profit organization. (2) The RECIPIENT of BIOLOGICAL RESOURCE shall obtain a prior written consent on use of it from the DEPOSITOR. (3) Any use of the BIOLOGICAL RESOURCE for profit purposes by a non-profit organization or any use of the BIOLOGICAL RESOURCE by a profit organization requires a separate license from the DEPOSITOR prior to distribution. (4) In publishing research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the original publication designated by the DEPOSITOR (Kaizuka, T. et al. Mol. Cell, 64 (4): 835-849, 2016) and an acknowledgment to the DEPOSITOR are requested. (5) The RECIPIENT must contact the DEPOSITOR in case of application for any patents or commercial use with the results from the use of BIOLOGICAL RESOURCE.
Additional terms and conditions:
Regarding pMRX vector: In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of literatures for pMX and pMRX vectors (Kitamura, T., Exp. Hematol. 31 (11): 1007-1014, 2003; Saitoh T., J. Biol. Chem. 278 (38): 36005-36012, 2003) is requested.

Remarks ((Additional form)) Approval Form(FormD)
Remember that you will be working with samples containing infectious virus.
This RESOURCE contains a fluorescent protein provided by Prof. Roger Tsien. UCSD's MTA is also required. Please visit Additional Forms section. This RESOURCE is not provided to for-profit organization or for for-profit research by non-profit organizations.
Please obtain written approval of the depositor.

提供案内 (日本国内) [open/close]

Ordering forms	Order form [Credit Card Payment] [Bank Transfer Payment] [Example of order form ] MTA, for use for not-for-profit academic purpose [Word] [Example of MTA ] MTA, for use for for-profit purpose [Word] Approval form by depositor [Word] Please visit Information of Request for Distribution.[link]
Terms and conditions for distribution	(1) BIOLOGICAL RESOURCE is limited to for academic research in non-profit organization. (2) The RECIPIENT of BIOLOGICAL RESOURCE shall obtain a prior written consent on use of it from the DEPOSITOR. (3) Any use of the BIOLOGICAL RESOURCE for profit purposes by a non-profit organization or any use of the BIOLOGICAL RESOURCE by a profit organization requires a separate license from the DEPOSITOR prior to distribution. (4) In publishing research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the original publication designated by the DEPOSITOR (Kaizuka, T. et al. Mol. Cell, 64 (4): 835-849, 2016) and an acknowledgment to the DEPOSITOR are requested. (5) The RECIPIENT must contact the DEPOSITOR in case of application for any patents or commercial use with the results from the use of BIOLOGICAL RESOURCE. Additional terms and conditions: Regarding pMRX vector: In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of literatures for pMX and pMRX vectors (Kitamura, T., Exp. Hematol. 31 (11): 1007-1014, 2003; Saitoh T., J. Biol. Chem. 278 (38): 36005-36012, 2003) is requested.
Remarks	((Additional form)) Approval Form(FormD) Remember that you will be working with samples containing infectious virus. This RESOURCE contains a fluorescent protein provided by Prof. Roger Tsien. UCSD's MTA is also required. Please visit Additional Forms section. This RESOURCE is not provided to for-profit organization or for for-profit research by non-profit organizations. Please obtain written approval of the depositor.

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] 寄託者による提供承諾書 [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	1. 非営利団体による学術研究に限る。2. 利用者は事前に寄託者の提供承諾書を得る。3. 当該リソースの非営利団体の営利目的での利用および営利団体の利用については、事前に寄託者との間で、別途使用許諾に係る契約の締結が必要である。4. 研究成果の公表にあたって寄託者の指定する文献(Kaizuka, T. et al. Mol. Cell, 64 (4): 835-849, 2016)を引用し、謝辞の表明を必要とする。5. 利用者が本件リソースを使用して得られた研究成果に基づき特許等の申請、及び事業活動を行う場合は、寄託者と別途協議を行う。付加的使用条件： pMRXベクターについて：利用者は、研究成果の公表にあたって pMXならびにpMRXベクターの文献（Kitamura, T., Exp. Hematol. 31 (11): 1007-1014, 2003; Saitoh T., J. Biol. Chem. 278 (38): 36005-36012, 2003) の引用を必要とする。
備考	【追加提供依頼書】承諾書(FormD) このリソースはウイルス粒子産生用のため、取扱いに注意が必要です。 Prof. Roger Tsienから供与された蛍光タンパク質を含みます。UCSDとのMTA締結が必要です。「申込みに必要な書式」をご覧ください。本件リソースは営利機関および非営利機関による営利目的研究には提供いたしません。寄託者の書面による提供承諾が必要です。

Catalog #	Resource name	Shipping form	Fee (non-profit org.)
RDB14601	pMRX-IP-GFP-LC3-RFP	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 pMRX-IP-GFP-LC3-RFP was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan (cat. RDB14601).

Reference section:

Morishita, H., Kaizuka, T., Hama, Y., Mizushima, N., A new probe to measure autophagic flux in vitro and in vivo. Autophagy 13 (4): 757-758 (2017). PMID 28121224. [link to RRC of NBRP]

Kaizuka, T., Morishita, H., Hama, Y., Tsukamoto, S., Matsui, T., Toyota, Y., Kodama, A., Ishihara, T., Mizushima, T., Mizushima, N., An Autophagic Flux Probe that Releases an Internal Control. Mol Cell. 2016 Nov 17;64(4):835-849 (2016). PMID 27818143. [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	Morishita, H., A new probe to measure autophagic flux in vitro and in vivo. Autophagy 13 (4): 757-758 (2017). PMID 28121224. [link to RRC of NBRP]
original	Kaizuka, T., An Autophagic Flux Probe that Releases an Internal Control. Mol Cell. 2016 Nov 17;64(4):835-849 (2016). PMID 27818143. [link to RRC of NBRP]
reference	Nguyen, T.B., DGAT1-Dependent Lipid Droplet Biogenesis Protects Mitochondrial Function during Starvation-Induced Autophagy. Dev. Cell 42 (1): 9-21 (2017). PMID 28697336. [link to Journal]
reference	Tamura, N., Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets. FEBS Lett. 591 (23): 3819-3830 (2017). PMID 29113029. [link to RRC of NBRP]
user_report	Ohnishi, K., Identification of Dietary Phytochemicals Capable of Enhancing the Autophagy Flux in HeLa and Caco-2 Human Cell Lines. Antioxidants (Basel) 9 (12): 1193 (2020). PMID 33261065. [link to RRC of NBRP]