Biosensor & Visualization

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Sensor & Visualization

• Autophagy indicator
  • Chaperone mediated autophagy activity by the GAPDH-HT indicator by Dr. Takahiro Seki’s lab as well as fluorescent protein probes of LC3B accumulation developed by Dr. Noboru Mizushima’s lab, Dr. Itaru Hamachi’s lab and Dr. Keiji Kimura’s lab are available.
• bilirubin indicator UnaG, BReleaCa
  • A dual-ligand-modulable fluorescent protein based on UnaG and calmodulin.
    Shitashima, Y., Shimozawa, T., Asahi, T., Miyawaki, A.
    Biochem. Biophys. Res. Commun. 496 (3): 872-879 (2018). PubMed PMID 29395087.

    Catalog no.	Name of resource	Descriptiion
    RDB15908	pRSETB-BReleaCa	Bacteria expression vector of UnaG/CaM hybrid protein, BReleaCa, capable of binding to bilirubin and calcium.
    Catalog no.	Name of resource	Descriptiion
    RDB15909	pcDNA3-BReleaCa	Expression vector of UnaG/CaM hybrid protein, BReleaCa, capable of binding to bilirubin and calcium.

  • Continuous de novo biosynthesis of haem and its rapid turnover to bilirubin are necessary for cytoprotection against cell damage.
    Takeda, T.A., Mu, A., Tai, T.T., Kitajima, S., Taketani, S.
    Sci. Rep. 5: 10488 (2015). PubMed PMID 25990790.

    Catalog no.	Name of resource	Descriptiion
    RDB13504	pET-His UNAG	Expression clone of eel UnaG in E.coli.

  • A bilirubin-inducible fluorescent protein from eel muscle.
    Kumagai, A., Ando, R., Miyatake, H., Greimel, P., Kobayashi, T., Hirabayashi, Y., Shimogori, T., Miyawaki, A.
    Cell 153 (7): 1602-1611(2013). PubMed PMID 23768684.

    Catalog no.	Name of resource	Descriptiion
    RDB15703	UnaG/pcDNA3-FLAG	Expression vector of eel UnaG, bilirubin-inducible fluorescent protein.
    Catalog no.	Name of resource	Descriptiion
    RDB15704	UnaG/pGEX-2T	xpression vector of eel UnaG, bilirubin-inducible fluorescent protein.

• cAMP indicator
  • Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells.
    Kitaguchi, T., Oya, M., Wada, Y., Tsuboi, T., Miyawaki, A.
    Biochem. J. 450 (2): 365-373 (2013). PubMed PMID 23282092.

    Catalog no.	Name of resource	Descriptiion
    RDB15247	Flamindo/pcDNA3	Expression vector of yellow fluorescent biosensor for cAMP, Flamindo.

• Cell cycle indicator Fucci
  • To monitor cell cycle progression in living cells, cell cycle indicator Fucci probes deveoped by Dr. Atsushi Miyawaki’s lab are available.
• Caspase activity indicator
  • A high-throughput method for development of FRET-based indicators for proteolysis.
    Nagai, T., Miyawaki, A.
    Biochem. Biophys. Res. Commun. 319 (1): 72-77 (2004). PubMed PMID 15158444.

    Catalog no.	Name of resource	Descriptiion
    RDB15705	SCAT3.1/pcDNA4 HMB	Expression vector of FRET based apoptosis probe.

  • Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer.
    Karasawa, S., Araki, T., Nagai, T., Mizuno, H., Miyawaki, A.
    Biochem. J. 381 (Pt 1): 307-312 (2004). PubMed PMID 15065984.

    Catalog no.	Name of resource	Descriptiion
    RDB15240	MiCy-DEVD-mKO1/pCS2	Expression vector of FRET based caspase3 activity monitoring probe.

• Calcium-ion sensor
  • Fluorescent protein-based Ca²⁺ sensors, G-CaMPs, Yellow Cameleons and Pericams developed by Dr. Jin-ichi Nakai’s lab and Dr. Atsushi Miyawaki’s lab, which are composed with calmodulin, fluorescent protein and M13 peptide (CaM binding domain of myosin light chain kinase), are designated to visualize intracellular [Ca²⁺] dynamics.
  • G-CaMP
  • Pericam
  • Yellow Cameleon
• Concentration of Protein in cells
  • Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it.
    Morikawa, J.T., Fujita, H., Kitamura, A., Horio, T., Yamamoto, J., Kinjo, M., Sasaki, A., Machiyama, H., Yoshizawa, K., Ichimura, T., Imada, K., Nagai, T., Watanabe, M.T.
    Sci. Rep. 6: 22342, 2016. PubMed PMID: 26956628

    Catalog no.	Name of resource	Descriptiion
    RDB14203	pPAL7_GimRET (CFP-YFP1G/pPAL7)	Expression vector of GimRET (Glycine inserted mutant FRET probe) for E. coli.
    RDB14204	pGimRET (CFP-YFP1G/pECFP)	Expression vector of GimRET (Glycine inserted mutant FRET probe) for mammalian cells.

• pH sensor
  • Dual-color-emitting green fluorescent protein used as pH indicators from the sea cactus Cavernularia obesa developed by Dr. Katsunori Ogoh of the Olympus corporation.
    Ogoh, K. et al. Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy. Luminescence 28 (4): 582-591, 2013.

    Catalog no.	Name of clone	Characteristic	misc.
    RDB14368	pCoGFP-wt	wild type
    RDB14369	pCoGFP-mam	From blue to green at pH 5 – 6
    RDB14370	pCoGFP-V0	From blue to green at pH 5 – 6
    RDB14371	pCoGFP-V1	From blue to green at pH 6 – 7
    RDB14372	pCoGFP-V2	From blue to green at pH 7 – 8
    RDB14373	pCoGFP-V3	From blue to green at pH 9 – 10
    RDB14374	pCoGFP-V4	From blue to green at pH 9 – 10

• RA indicator
  • Visualization of an endogenous retinoic acid gradient across embryonic development.
    Shimozono, S., Iimura, T., Kitaguchi, T., Higashijima, S., Miyawaki, A.
    Nature 496 (7445): 363-366 (2013). PubMed PMID 23563268.

    Catalog no.	Name of resource	Descriptiion
    RDB15249	GEPRA-B	Genetically encoded indicator for RA, GEPRA, having ligand binding domain from the RAR-beta.
    RDB15250	GEPRA-G	Genetically encoded indicator for RA, GEPRA, having ligand binding domain from the RAR-gamma.
    RDB15251	GEPRA-AA	Genetically encoded indicator for RA, GEPRA. Low-affinity verson of the GEPRA-B.
    RDB15252	GEPRA-B/ pT2KXIGdeltain	Expression vector of genetically encoded indicator for RA, GEPRA. For generation of transgenic zebrafish.
    RDB15253	GEPRA-G/ pT2KXIGdeltain	Expression vector of genetically encoded indicator for RA, GEPRA. For generation of transgenic zebrafish.
    RDB15254	GEPRA-AA/ pT2KXIGdeltain	Expression vector of genetically encoded indicator for RA, GEPRA. For generation of transgenic zebrafish. Low-affinity verson of the GEPRA-B.

• Sphingolipid marker
  • Lipid rafts are small lipid domains on the cell membrane and are thought to play an important role in signal transduction, endocytosis and more. We provide fluorescent probes for sphingomyelin and cholesterol lipid domains.
  • Nakanori: sphingomyelin and cholesterol lipid domain (lipid raft)
  • D4 toxin: cholesterol rich domain
  • lysenin: sphingomyelin
• Voltage indicator
  • Improving membrane voltage measurements using FRET with new fluorescent proteins.
    Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A.
    Nat. Methods 5 (8): 683-685 (2008). PubMed PMID 18622396.

    Catalog no.	Name of resource	Descriptiion
    RDB15262	Mermaid	Voltage indicator Mermaid.

  • Improved detection of electrical activity with a voltage probe based on a voltage-sensing phosphatase.
    Tsutsui, H., Jinno, Y., Tomita, A., Niino, Y., Yamada, Y., Mikoshiba, K., Miyawaki, A., Okamura, Y.
    J. Physiol. 591 (18): 4427-4437 (2013). PubMed PMID 23836686.

    Catalog no.	Name of resource	Descriptiion
    RDB15263	Mermaid2	Voltage probe Mermaid2.

• Notch signaling reporter
  • The pRBS-EGFP and RBP-J-Venus expression clones deposited by Dr. Makoto Mark Taketo and Dr. Kenji Tanigaki, respectively, allow you monitoring the state of activation of the Notch signaling by fluorescence in living cells.
• Epigenetics reporter
  • Visualization of histone acetylation: The Histac fluorescent probes deposited by Dr. Kazuki Sasaki allow you monitoring the state of activity of acetylation of histone H4 by fluorescence in living cells.
  • Visualization of methylated DNA: The EGFP-MBD-nls protein recognizes the methylated DNA and you can follow status of the DNA methylation in situ under physiological conditions using the pEGFP-MBD-nls expression clone.
• Organelle marker/subcellular localization
  • We are providing genetic resources for visualization of organelles such as mitochondria and nucleus. Each clone contains an organelle localization signal sequence fused with fluorescent proteins or epitope tags. Organelles can be detected by fluorescence or by detecting epitope tags with antibodies.
• Fluorescent protein probe that can visualize the multiple inter-organelle contact sites in cells.
  • Divided GFP-fragment have a feature that they emit fluorescence again by reconstitution when each fragment is in close proximity. When labelled organelles such as mitochondria and ERs by the divided GFPs exist together, the contact sites in cells are visualized by the reconstituted fluorescent protein. The vectors of fluorescent protein probes developed by Dr. Yasushi Tamura of the Yamagata University, Dr. Toshiya Endo of the Kyoto Sangyo University and their colleague are available from the DNA Bank.
    
  • Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.
    Kakimoto, Y., Tashiro, S., Kojima, R., Morozumi, Y., Endo, T., Tamura, Y.
    Sci. Rep. 8 (1): 6175 (2018). PubMed PMID 29670150.

    Catalog no.	Name of resource	Descriptiion
    RDB16037	Tom71-GFP1-10	Expression vector of S. cerevisiae protein channel TOM71 (Tom 71) for visualizing organelle contact sites in vivo.
    RDB16038	Tom71-GFP11	Expression vector of S. cerevisiae protein channel TOM71 (Tom 71) for visualizing organelle contact sites in vivo.
    RDB16039	Tom71-V5-GFP11	Expression vector of S. cerevisiae protein channel TOM71 (Tom 71) for visualizing organelle contact sites in vivo.
    RDB16040	YNR021W-GFP11	Expression vector of S. cerevisiae endoplasmic reticulum (ER) protein YNR021W (hypothetical protein) for visualizing organelle contact sites in vivo.
    RDB16041	Tom20N-FLAG-GFP1-10	Expression vector of human translocase of outer mitochondrial membrane 20 (Tom20), N-terminal segments 1-33 for visualizing organelle contact sites in vivo.
    RDB16042	Tom70N-FLAG-GFP1-10	Expression vector of human translocase of outer mitochondrial membrane 70 (Tom70), N-terminal segments 1-70v
    RDB16043	Ifa38-GFP1-10	Expression vector of S. cerevisiae ketoreductase (Ifa38) for visualizing organelle contact sites in vivo.
    RDB16044	Ifa38-GFP11	Expression vector of S. cerevisiae ketoreductase (Ifa38) for visualizing organelle contact sites in vivo.
    RDB16045	Ifa38-FLAG-GFP11	Expression vector of S. cerevisiae ketoreductase (Ifa38) for visualizing organelle contact sites in vivo.
    RDB16046	Ifa38-mCherry-GFP11	Expression vector of S. cerevisiae ketoreductase (Ifa38) for visualizing organelle contact sites in vivo.
    RDB16047	ERj1N-V5-GFP11	Expression vector of human DnaJ Heat Shock Protein Family (Hsp40) Member C1, N-terminal segments 1-200 for visualizing organelle contact sites in vivo.
    RDB16048	Sec63N-V5-GFP11	Expression vector of human SEC63 homolog, protein translocation regulator (Sec63), N-terminal segments 1-240 for visualizing organelle contact sites in vivo.
    RDB16049	Vph1-GFP1-10	Expression vector of S. cerevisiae H(+)-transporting V0 sector ATPase subunit a (Vph1) for visualizing organelle contact sites in vivo.
    RDB16050	Dpp1-V5-GFP11	Expression vector of S. cerevisiae bifunctional diacylglycerol diphosphate phosphatase/phosphatidate phosphatase (Dpp1) for visualizing organelle contact sites in vivo.
    RDB16051	Pex3N-GFP1-10	Expression vector of S. cerevisiae Pex3p (Pex3), N-terminal segments 1-60 for visualizing organelle contact sites in vivo.
    RDB16052	Pex3N-FLAG-GFP11	Expression vector of S. cerevisiae Pex3p (Pex3), N-terminal segments 1-60 for visualizing organelle contact sites in vivo.
    RDB16053	Erg6-GFP1-10	Expression vector of S. cerevisiae sterol 24-C-methyltransferase (Erg6) for visualizing organelle contact sites in vivo.
    RDB16054	Erg6-FLAG-GFP11	Expression vector of S. cerevisiae sterol 24-C-methyltransferase (Erg6) for visualizing organelle contact sites in vivo.

References

Fluorescent protein resource

• Sea cactus therostable GFP
• Azami Green (AG, hmAG1, hmAG407)
• cjBlue (cjBlue, cjBlue Y64L)
• Cy11.5
• Dronpa-Green (Dronpa, Dronpa2, Dronpa3, 22G)
• Kaede
• Keima-red (dKeima, dKeima570, mKeima, tdKeima)
• Kikume Green-Red (KikGR, mKikGR, mKikGR13.2, Xpa)
• Kusabira Green Orange (mK-GO)
• Kusabira Orange (KO1, hmKO1, hmKO2, hmKO-K)
• Midoriishi-Cyan (MiCy, mMiCy1)
• Venus (Venus, mVenus, cp49Venus, cp145Venus, cp157Venus, cp173Venus, cp195Venus, cp229Venus)
• Destabilized FPs
• Knock in markers with CRISPR/Cas9 genome editing

Luminescent protein

• Nano-lantern luminescent and fluorescent protein
  • Nano lantern (NL) consists of Renilla luciferase and adjacent fluorescence protein. The chemiluminescence of the luciferase provides light source for excitation and enables the fluorescence protein to be observed. Three colors of NLs, yellow, cyan and orange, have been developed. NLs do not require external light source and overcome problems such as autofluorescence, phototoxicity, and photobleaching.
• Higher intensity luciferases having Green-, Yellow- or Red-emission by using D-luciferin
  • These luciferases have at most four-time maximum luminous intensity than that of widely used luciferase of the Photinus pyralis, a common North American firefly. Further more, Green-, Yellow- or Red-emission can be obtained by using D-luciferin as a substrate of each luciferase.
• AkaLuc luciferase providing brighter and red-shifted luminescence
  • The artificial bioluminescence system AkaBLI enables noninvasive signal observation in deep tissue of living animals. It was developed by Dr. Atsushi Miyawaki and Dr. Satoshi Iwano of the RIKEN Center for Brain Science, and Dr. Shojiro Maki of the University of Electro-Communications. The AkaBLI consists of an artificial substrate AkaLumine with improved tissue permeability and an artificial luciferase Akaluc optimized to AkaLumine. The intensity of the luminescence of AkaBLI system is 100 to 1000 folds brighter than the conventional systems.

(GRP0058e 2019.01.24 T.M.)

2019.04.24