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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

Dnaconda’s Recommendation


dnaconda01

 

HMGA1 influences urokinase plasminogen activator system throught the regulation of PLAU and SERPINE1 gene. (2019/4/12 T.M.)

  • HMGA1 is a oncofetal architectural transcription factor and has a role in controling genes in many of aspects of neoplastic transformation.
  • The authors revailed secreted proteins whose abundance was linked to HMGA1 expression level. Among them, it is demonstrated that HMGA1 has a positive modulation function on the regulatory elements of PLAU and SERPINE1 genes, well known components of the urokinase plasminogen activator system that has a prominent role in promoting metastasys. pGL4-phPLAU (RDB07487) and pGL4-phSERPINE1(PAI-1) (RDB07461) were used for the reporter assay.
  • Reference
    Resmini, G., Rizzo, S., Franchin, C., Zanin, R., Penzo, C., Pegoraro, S., Ciani, Y., Piazza, S., Arrigoni, G., Sgarra, R., Manfioletti, G. HMGA1 regulates the Plasminogen activation system in the secretome of breast cancer cells. Sci. Rep. 7 (1): 11768 (2017). PMID: 28924209.
  • Plssmid clone
    pGL4-phPLAU (cat# RDB07487)
    pGL4-phSERPINE1(PAI-1) (cat# RDB07461)


Fluorescent protein probe that can visualize the multiple inter-organelle contact sites in cells. (2019/3/29 N.N.)

  • 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.
  • Reference
    Kakimoto, Y et al. Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system. Sci. Rep., 8 (1): 6175, 2018. PMID : 29670150.
  • DNA resource
    Please visit the list of resources deposited by Dr. Tamura.[link]


Recombinant protein expression vector in fruit fly cell line, S2. (2019/2/22 T.Y.)

  • The Drosophila culture cell (S2) is widely used for recombinant mammalian proteins, because of more chance to get active protein than bacteria and more chance to get free from interactors than mammalian culture cells. Here, authors introduce three additional tools (see below) for original pMT-PURO (cat# RDB08532) to aim more effective transfection and more stable transfectant during cultivation.
  • 1. original pMT-PURO (cat# RDB08532): Containing methallothionein promoter, which activated by CuSO4 (~0.5 mM final conc.) added to culture medium, followed by cloning site, and “puromycin resistant” gene pac (puromycin N-acetyl transferase) as well.
  • 2. pMT-PURO2 (cat# RDB12152): The Kozak consensus sequence is inserted to the upstream of pac gene on pMT-PURO to aim highly stable pac expression. Thus, transfectant of pMT-PURO2 should be more stable during cultivation than that of original pMT-PURO.
  • 3. pMT-PURO2G (cat# RDB09004), pMT-PURO2R (cat# RDB09005): EGFP and DsRed2 are respectively fused to pac on pMTPURO2 (cat# RDB12152) to visualize transfectant. Thus, you can explore to find effective transfection condition of the observation of the fluorescence before scale up the cultivation.
  • Reference
    Nagahashi, K. et al. Fusion of fluorescent protein to puromycin N-acetyltransferase is useful in Drosophila Schneider S2 cells expressing heterologous proteins. Cytotechnology. 65 (2): 173-178, 2013. PMID : 22706964.
  • DNA resource
    pMT-PURO (cat# RDB08532)
    pMT-PURO2 (cat# RDB12152)
    pMT-PURO2G (cat# RDB09004)
    pMT-PURO2R (cat# RDB09005)


Quantitative evaluation of autophagic activity(2019/2/1 N.N.)

  • In order to visualize autophagosome and observe autophagy, MAP1LC3 (LC3) fused with fluorescent proteins as a probe are used. However, the fluorescence of the probe attenuates during autophagy progresses and it is not suitable for quantitative assay of autophagy.
  • Dr. Noboru Mizushima and his colleagues at the University of Tokyo developed a new probe that can expresses GFP-LC3 and RFP-LC3deltaG (lacking glycine (G) at the LC 3 end) simultaneously at equimolar amounts.
  • As autophagy progresses, the fluorescence of GFP-LC3 decays whereas that of RFP-LC3deltaG remains in the cytoplasm as an internal standard. Therefor, the activity of autophagy can be quantified evaluated by the fluorescence intensity ratio of GFP/RFP.
  • Using this probe, autophagic flux was measured in mouse and zebrafish embryos and tissues as well as cultured cells. This is also suitable for analysis of large number of samples.
  • Reference
    Kaizuka, T. et al. An Autophagic Flux Probe that Releases an Internal Control. Mol. Cell, 64 (4): 835-849, 2016. PMID: 27818143
  • Plssmid clone
    pMRX-IP-GFP-LC3-RFP-LC3 delta G (cat# RDB14600)


Expression vector of optogenetics technique (2019/1/11 N.N.)

  • Expression vector of the channelrhodopsin-green receiver (ChRGR) (cat# RDB16748) which induces membrane potential oscillation by green light irradiation.
  • Expression vector of the ChRGR(ER) (cat# RDB16750) which localizes ChRGR to endoplasmic/sarcoplasmic reticulum (ER/SR). ChRGR(ER) is used as a tool to manipulation of intracellular calcium ion release.
  • Reference
    Asano, T. Igarashi, H. Ishizuka, T. Yawo, H. Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light. Front. Neurosci. 12 :561, 2018. PMID: 30174581.
  • DNA resource
    pCAGGS-ChRGR-Venus (cat# RDB16748)
    pCAGGS-ChRGR(ER)-Venus (cat# RDB16750)
    Further Genetic Resources for Optgenetics Research


mCherry expression vector with Cre/loxP system (2018/12/7 S.H.)

  • Dr. Yumiko Hatanaka and her colleagues at Osaka University developed mCherry expression vector with Cre/loxP system. In the original paper, lateral cortical VZ cells of mouse embryo at E12.5 were labeled with mCherry by in utero electroporation, using mixture of pCALNL5:mCherry and about 10,000 lower concentration of Cre recombinase expression vector. The authors analyzed progeny cells in brain preparation and revealed their dynamics by means of classification with their axon-like processes length and number.
  • Reference
    Hatanaka, Y. and Yamauchi, K. Excitatory Cortical Neurons with Multipolar Shape Establish Neuronal Polarity by Forming a Tangentially Oriented Axon in the Intermediate Zone. Cerebral Cortex J., 23: 105, 2013. PMID 22267309.
  • DNA resource
    pCALNL5:mCherry (cat# RDB16747)
  • Cre recombinase expression vectors [link] are available from the DNA Bank.

Genetic resources to visualize organelle (2018/11/14 N.N.)

  • We are providing genetic resources for visualization of 15 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.
  • Of those, 12 species of organelles can be observed by Nano-lanterns developed by Drs. Okada and Takai of the RIKEN Center for Biosystems Dynamics Research (BDR) and Nagai and their colleague of the Osaka University [Takai, A. et al., Proc Natl Acad Sci U S A. 112 (14): 4352-4356, 2015]. Nano-lantern is a brighter luminescent protein, consisting of luciferase and fluorescent protein. Three colors (yellow, cyan and orange) of Nano-lanterns are available for each localization signal. We are looking forward to receiving your request.
  • List of organelles
  • List of Nano-lanterns


Akaluc luciferase providing brighter and red-shifted luminescence (2018/5/7 N.N.)

  • Firefly bioluminescence systems have been commonly used as a imaging tool of biological phenomena. However, it have not been strong enough for imaging signals in deep tissues, because of low permeabilization of substrates. Recently, Dr. Atsushi Miyawaki and Dr. Satoshi Iwano of the RIKEN Center for Brain Science, and Dr. Shojiro Maki of the University of Electro-Communications developed a system of artificial bioluminescence AkaBLI that enables noninvasive signal observation in deep tissue of living animals.
    The AkaBLI system 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.
    The expression vector of artificial luciferase Akaluc has been deposited by Dr. Miyawaki’s lab and it is now available from the DNA Bank.
  • Reference
    Iwano, S. et al., Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 359 (6378): 935-939 (2018). PMID 29472486.
  • Press Release and Blog
    In living color: seeing cells from outside the body with synthetic bioluminescence (2018/2/23, RIKEN)
    In living color: imaging the brain with synthetic bioluminescence (2018/2/23, It Ain’t Magic Blog)
  • DNA resource
    pcDNA3 Venus-Akaluc (cat# RDB15781)


Genome analysis of metabolic pathways between syntrophic bacteria. (2018/3/2 K.N.)

  • Degradation of branched-chain fatty acids (BCFAs) via the syntrophic association of bacteria is an essential step in the production of methane from proteins/amino acids in anaerobic environment. In order to elucidate the metabolic pathways in the syntrophic bacteria, a comparative genome analysis was performed between BCFA-degradable and non degradable strains. The genomic DNA of the JCM 14075T strain was used as BCFA degradable strain standard.
  • Reference
    Narihiro, T. et al. Comparative Genomics of Syntrophic Branched-Chain Fatty Acid Degrading Bacteria. Microbes. Environ. 31 (3): 288-922 (2016). PMID 27431485.
  • DNA Resource
    Genomic DNA of Syntrophomonas wolfei subsp. methylbutyratica JCM 14075T (cat# JGD12758)


Stable ChIP-seq analysis using transcription factors tagged with biotinylated peptide tag. (2018/2/16 K.N.)

  • ChIP-seq is a powerful method of comprehensive analysis of transcription factor (TF) binding sites. However, the bottle neck of ChIP-seq analysis is the quality of antibody, and thus many researchers have been given up. To overcome, authors constructed the vector expressing “biotinylated-tag” transcription factors. With this vector, you can get acceptable level of TF-DNA complex for ChIP-seq analysis by streptavidin-beads for any kinds of TF.
  • Reference
    Matsuda, K. et al., ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network. Development 144 (11): 1948-1958, 2017. [PMID 28455373]
  • DNA Resource
    pCAGGS-BLRP-BirA (cat# RDB15774)


Fluorescent probes for sphingomyelin and cholesterol lipid domains.(2018/02/06 N.N.)

  • Lipid rafts are small lipid domains on the cell membrane and are thought to play an important role in signal transduction, endocytosis and more. Because of the lack of appropriate probes to label sphingomyelin and cholesterol individually, detailed analysis of lipid rafts was difficult.
  • Dr. Toshihide Kobayashi and his colleagues of the former Lipid Biology Laboratory at RIKEN have developed methods to visualize the lipid rafts using probes of lipid binding proteins fused with fluorescent proteins.
  • DNA Bank provides plasmids of the mCherry and GFP fluorescent probes that can label lipid rafts, sphingomyelin and cholesterol.We are looking forward to receiving your request soon.
  • Reference
    Genes involved in the sphingolipid signaling pathway [link]
    Makino, A. et al. FASEB J., 31 (4): 1301-1322, 2017. [PMID: 27492925]
    Makino, A. et al. FASEB J., 29 (2): 477-493, 2015. [PMID: 25389132]
    Shimada, Y. et al. Eur. J. Biochem., 269 (24): 6195-6203, 2002. [PMID: 12473115]


Establishment of Gpr151-Cre mouse using a BAC clone and disruption of selected mature neurons. (2018/2/5 T.M.)

  • MSMg01-081G04 was used to construct BAC clone having NLS-Cre expression cassette under the control of Gpr151 promoter and Gpr151-Cre mouse was generated. Combined with Cre-specific lacZ reporter mouse, the targeted cells possessed in cholinergic neurons of the medial nucleus of habenular complex (mHb) projecting to the interpeduncular nucleus (IPN). The Cre-expressing mouse was used to disrupt selected neurons by the combination with Eno2-Diphtheria toxin A mice.
  • Reference
    Kobayashi Y. et al., Genetic dissection of medial habenula-interpeduncular nucleus pathway function in mice. Front Behav. Neurosci. 7: 17 (2013). PMID: 23487260.
  • DNA Resource
    MSM/Ms Mouse BAC clone
    MSM/Ms mouse BAC references
    MSMg01-081G04 (searching result)


Analysis of bacterial cytoskeleton of Bacillus anthracis in fission yeast. (2018/2/5 T.M.)

  • It is known that bacterial cytoskeleton is use by pathogenic prokaryote to segregate virulence plasmids. Bacillus anthracis TubZ (Ba-TubZ) participates pXO1 segregation but its biophysical properties have not been fully explored. Understanding the properties of Ba-TubZ, GFP fusion proteins are expressed in fission yeast and the microscopic images observed by a confocal microscope are analyzed.
  • Reference
    Srinivasan, R., Mishra, M., Leong, F.Y., Chiam, K.H., Balasubramanian, M. Bacillus anthracis tubulin-related protein Ba-TubZ assembles force-generating polymers. Cytoskeleton (Hoboken) 68 (9): 501-511 (2011). PMID 21780309.
  • DNA Resource
    pDUAL-HFG1c (cat# RDB06133)
    pDUAL-HFG41c (cat# RDB06134)
    pHFG41-ccdB2 (cat# RDB06526)


Hedgehog signal analysis by the Gli recognition element reporter. (2018/2/5 T.M.)

  • To elucidate roles of oxidative stress during the induction of osteoporosis and inhibition of hedgehog signaling, osteoblast differentiation in murine embryonic mesenchymal cell C3H10T1/2 was investigated with a focus on the interaction with mitogen-activated protein kinase (MAPKs) as possible mediator. To monitor Gli-mediated transcription activity in C3H10T1/2 cells under the oxidative stress and application of MAPK inhibitors, luciferase reporter having Gli-transcription factor recognition element was used and the inhibitory effect of JNK1 on Gli transcription was suggested.
  • Reference
    Shiohama, T., Fujii, K., Uchikawa, H., Takatani, T., Mizuochi, H., Kohno, Y. Oxidative stress-induced JNK1 phosphorylation inhibits hedgehog signalling and osteoblast differentiation. Cell Biol. Int. Rep. 21 (2): 53-62 (2014).
  • DNA Resource
    8×3’Gli-BS-delta51-LucII (cat# RDB08061)
    8xm3’Gli-BS-delta51-LucII (cat# RDB08062)
    pcDNA3.1-His-Gli1 (cat# RDB08064)


Genes promoting hepatocyte differentiation from human iPS cells. (2018/2/5 T.M.)

  • Expression profile of genes was compared between human adult liver and 201B7 cells (iPS cells). Sixteen genes were identified as initial candidates and these expression vectors were constructed. To identify genes promoting hepatocyte differentiation from iPS cells, 210B7 cells ware transfected with these expression vector to screen genes that upregulate alpha-feto protein (AFP) and downregulate Nanog and identified CEBPA, CEBPB, FOXA1 and FOXA3 genes.
    Induction of AFP and albumin were stronger in 201B7 cells transfected with CEBPA, CEBPB, or the combination of the four genes than in untransfected cells and these results suggested these four genes were suitable for the induction of hepatocyte differentiation from iPS cells.
  • Reference
    Tomizawa, M., Shinozaki, F., Motoyoshi, Y., Sugiyama, T., Yamamoto, S., Ishige, N. Transcription Factors and Medium Suitable for Initiating the Differentiation of Human-Induced Pluripotent Stem Cells to the Hepatocyte Lineage. J. Cell. Biochem. 117 (9): 2001-2009 (2016). Pubmed ID: 26773721.
  • DNA Resource
    Genome Network Project Clone
    IRCB003O22 (cat# HGY121558)
    IRAK047O19 (cat# HGY019155)
    IRAK015C16 (cat# HGX006064)
    IRAL031O05 (cat# HGY092741)
    IRAK048J15 (cat# HGY019431)
    IRAK027I02 (cat# HGX010994)
    W01A073N10 (cat# HGE029522)
    NRCD Human cDNA Clone
    Are07G01 (cat# HKR042945).


Promotion of steroidogenic cell differentiation by SF-1 gene expression. (2018/2/5 T.M.)

  • In 2012, the authors reported a method to induce steroidogenic cells from the human iPS cell-derived mesodermal cells using the SF-1 expression clone pCMFlag_hsNR5A1 (RDB06299) (Sonoyama et al., 2012). Now, they studied a role of dopamine D1 receptor signaling mediated-induction of steroidogenic cells from the iPS cell-derived early intermediate mesoderm.
  • Reference
    Matsuo, K. et al., Significance of dopamine D1 receptor signaling for steroidogenic differentiation of human induced pluripotent stem cells. Sci. Rep. 7 (1): 15120 (2017). PMID: 29123220.
    Sonoyama, T. et al., Differentiation of human embryonic stem cells and human induced pluripotent stem cells into steroid-producing cells. Endocrinology 153 (9): 4336-4345 (2012). PMID: 22778223.
  • DNA Resource
    pCMFlag_hsNR5A1 (cat# RDB06299)


ATF6-dependent upregulation of SESN2 by ER stress. (2018/2/5 T.M.)

  • Growing evidence suggest SESN2 is upregulated by various stress and promotes cellular adaptation against stress. In the study of ATF6-dependent induction of SESN2 by ER stress pGL4-phSESN2 (RDB07413) was applied with ATF6 inhibition by forced expression of dominant negative ATF6 alpha or siRNA.
  • Reference
    Jegal, K.H. et al., Activating transcription factor 6-dependent sestrin 2 induction ameliorates ER stress-mediated liver injury. Biochim. Biophys. Acta 1864 (7): 1295-1307 (2017). PMID 28433684.
  • DNA Resource
    pGL4-phSESN2 (cat# RDB07413)


Fluorescent Ubiquitination-based Cell Cycle Indicator Fucci expression plasmid (2017/11/15 N.N.)

  • Dr. Atsushi Miyawaki and his colleagues at RIKEN Brain Science Institute (RIKEN BSI) developed Fluorescent Ubiquitination-based Cell Cycle Indicators, Fucci and Fucci2 that allow real-time observation of cell cycle.
  • DNABank provides Fucci2 plasmids for transfection into cells, for targeting into mouse R26 locus, and for establishment of zebrafish strains. Mouse strains and cell lines containing Fucci or Fucci2 are also available from the RIKEN BRC.
  • Reference :
    Fucci – Fluorescent Ubiquitination-based Cell Cycle Indicator [link]
    A window to embryonic development (RIKEN, 2010) [link]
    The color and the shape (RIKEN, 2009) [link]
    Waiting for the light to change (RIKEN, 2008) [link]
    Sakaue-Sawano, A. et al. Mol. Cell 68 (3): 626-640.e5, 2017. [PMID: 29107535]
    Mort, R.L. et al. Cell Cycle 13 (17): 2681-2696 ,2014. [PMID: 25486356]
    Sakaue-Sawano, A. et al. BMC. Cell Biol. 12: 2, 2011. [PMID: 21226962]
    Sugiyama, M. et al. Proc. Natl. Acad. Sci. USA. 106 (49): 20812-20817, 2009. [PMID: 19923430]
  • DNA Resource
    Fucci – Fluorescent Ubiquitination-based Cell Cycle Indicator


Distribution of Host E. coli strains for incorporation of synthetic amino acids.(2017/07/25 N.N.)

  • The host E. coli strains, RFzero-iy strain and B-95.deltaA strain that can produce recombinant proteins incorporating synthetic amino acids by reassignment of the UAG codon.
  • The RFzero-iy strains are designated to incorporate 3-iodotyrosine into produced proteins. For the incorporation, the strain is transformed with the expression plasmid in which a codon for the target tyrosine is replaced with UAG codon and cultured in 3-iodotyrosine containing medium (Mukai, T. et al., 2011). Two strains based on the BW25113 and BL-21(DE3) are available.
  • The B-95.deltaA strains can be assigned their UAG codon to synthetic amino acids by the introduction of plasmids carrying specific pair of UAG-reading tRNA and an aminoacyl-tRNA synthetase (aaRS) variant*. Increased productivity of sulfonated hirudin which is expected to improve the inhibition of blood coagulation was reported (Mukai, T. et al., 2015). The original and the growth improved derivative strains are available.
  • Reference
    Mukai, T. et al., Biochem. Biophys. Res. Commun. 411 (4): 757-761, 2011. [PMID 21782790]
    Mukai, T. et al., Sci. Rep. 5: 9699, 2015. [PMID 25982672]
    Incorporation of synthetic amino acids into proteins at specific sites
  • DNA Resource
    BW25113-based RFzero-iy (cat # RDB14427)
    BL21(DE3)-based RFzero-iy (cat # RDB14428)
    B-95.deltaA (cat # RDB13711)
    B-95.deltaAdeltafabR (cat # RDB13712)
    *Plamids that introduce synthetic amino acids are provided by RIKEN CLST.


Evaluation of chaperone mediated autophagy (CMA) activity by the GAPDH-HT indicator. (2017/06/29 N.N.)

  • “GAPDH-HT indicator” is a CMA marker consisting of HaloTag and GAPDH which is a typical substrate of CMA. The CMA activity in the cells can be evaluated by using GAPDH-HT indicator and an appropriate under the fluorescent-labeled HaloTag ligand.
  • Reference
    Seki, T. et al. Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron. PLoS One 7 (2): e31232, 2012. PMID: 22363588
    Sato, M. et al. Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells. Genes Cells 21 (8): 861-873, 2016. PMID: 27377049
  • DNA Resource
    GAPDH-HT/pcDNA5/FRT (cat # RDB15088)


Establishment of deficient cells using Cre recombinase and floxed mouse derived cells (2017/06/23 T.M.)

  • Has2 deficient cells were established by using Cre recombinase expressing AxCANCre adenovirus. Mouse Has2 gene floxed allele cells were segregated from the transgenic mice having the exon 2 flanked by two loxP sites. And then the cells were infected with AxCANCre adenovirus carrying the Cre recombinase to remove the exon 2.
  • Reference
    Chanmee, T. et al. Hyaluronan production regulates metabolic and cancer stem-like properties of breast cancer cells via hexosamine biosynthetic pathway-coupled HIF-1 signaling. J. Biol. Chem. 291 (46): 24105-24120, 2016. PMID: 27758869.
  • DNA Resource
    AxCANCre (cat# RDB01748)


M-INK, a novel tool for tagging the mature melanosomes.(2017/05/02 N.N.)


G-CaMP, a fluorescent calcium sensor.(2017/03/16 N.N.)

  • A Fluorescent probes “G-CaMP” for measuring intracellular calcium concentration are available.
  • The G-CaMPs are calcium sensor proteins consisting of Calmodulin (CaM) calcium-binding region, M13 fragment of myosin light chain kinase and fluorescent protein. In the original paper, G-CaMPs were used to observe cardiomyocytes differentiated from iPS cells (Shiba, Y. et al, 2016) and neurons of zebrafish (Muto, A. et al, 2011).
  • Reference
    • Shiba, Y. et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature, 538 (7625): 388-391, 2016. PMID: 27723741
    • Ohkura, M. et al. Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. PLoS One, 7 (12): e51286, 2012. PMID: 23240011
    • Ohkura, M. et al. An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials. PLoS One, 7 (7): e39933, 2012. PMID: 22808076
    • Muto, A. et al. Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. Proc. Natl. Acad. Sci. U S A., 108 (13): 5425-5430, 2011. PMID: 21383146
  • DNA Resource
    G-CaMP4.1 (cat.# RDB14606)
    G-CaMP-HS (cat.# RDB14607)
    G-CaMP6 (cat.# RDB14609)
    G-CaMP7 (cat.# RDB14610)
    G-CaMP8 (cat.# RDB14611)
    G-CaMP7.09 (cat.# RDB14612)

    Please also visit Calcium Ion Sensor Clones.


Construction of reporter gene having long promoter by using BAC clone and with recombineering technique.(2017/02/28 T.M.)

  • Luciferase reporter assay is a powerful tool for real-time monitoring of gene expression in living cells. It is recommended to construct reporter gene with lager promoter but hard to do so by means of common cloning technique. In this article, 20 kb promoter of Hprt gene was retrieved and cloned into a luciferase reporter by using BAC clone and with recombineering technique.
  • Reference
    Endo, T. et al. Evaluation of an Hprt-luciferase reporter gene on a mammalian artificial chromosome in response to cytotoxicity. Yonago Acta Med. 59 (2): 174-182 PMID: 27493490.
  • DNA Resource
    C57BL/6N (B6N) mouse BAC clone
    C57BL/6N (B6N) mouse BAC references
    Mouse B6N BAC clone B6Ng01-126E09 (Clone search results)


A fluorescent probe to evaluate activity of autophagy. (2017/02/21 N.N.)

  • Plasmid clone of a novel probe capable of evaluating the activity of autophagy by GFP/RFP signal ratio has arrived.
  • Autophagy in the mouse embryonic fibroblasts, HeLa cells and the zebrafish embryo was observed in the original paper.
  • Reference
    Kaizuka, T. et al. An Autophagic Flux Probe that Releases an Internal Control. Mol. Cell, 64 (4): 835-849, 2016. PMID: 27818143.
  • Deposited ResourcepMRX-IP-GFP-LC3-RFP-LC3deltaG (catalog#RDB14600)
    pMRX-IP-GFP-LC3-RFP (catalog#RDB14601)


Green- and Red-emitting luciferases with luminescence.(2017/2/2 T.M.)


Histac-H3K9/K14 for visualization of acetylation activity of histone H3 in cells.(2016/9/27 T.M.)

  • Reference
    Nakaoka, S., Sasaki, K., Ito, A., Nakao, Y., Yoshida, M. A Genetically Encoded FRET Probe to Detect Intranucleosomal Histone H3K9 or H3K14 Acetylation Using BRD4, a BET Family Member. ACS Chem. Biol. 11 (3): 729-733, 2016. PMID: 25946208
  • DNA Resource
    1. pcDNA3.1(+)-Histac-H3K9/K14 (catalog#RDB14340)


GimRET for visualization of protein concentration in cells.(2016/9/13 T.M.)


Nakanori that specifically binds to a complex of sphingomyelin and cholesterol.(2016/9/13 T.M.)


Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci) by single plasmid.(2016/6/20 T.M.)

  • Fucci2a cell cycle phase markers allow visualization and estimation of cell cycle progress by observation of green and red fluorescent proteins. This plasmid express two distinct colored fluorescent proteins, one of which fused to Cdt1 degron, another one of which fused to Geminin degron. Therefore, the transfectant indicate distinct color fluorescence between the cell (G1) and the cell (S/G2/ M).
  • Reference
    Mort, R.L., Ford, M.J., Sakaue-Sawano, A., Lindstrom, N.O., Casadio, A., Douglas, A.T., Keighren, M.A., Hohenstein, P., Miyawaki, A., Jackson, I.J. Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice. Cell Cycle 13 (17): 2681-2696, 2014.
  • DNA Resource
    1. pCAG-Fucci2a (catalog#RDB13080) for transient expression of Fucci2a marker.
    2. pROSA-floxNeo-Fucci2a (catalog#RDB13081) for targeted insertion of Fucci2a marker into mouse ROSA26 locus.

    Transgenic mouse B6;129-Gt(ROSA)26Sor (catalog # RBRC06511) is also available from the Experimental Animal Division.


Mutation analyses of genes on 6p12-p11 in patients with juvenile myoclonic epilepsy. (2016/4/12 K.N.)

  • Authors had been narrowed down to 3.5 cM size on a region of short arm of chromosome 6 (6p12-p11) as the genomic region (EJM1) related to juvenile myoclonic epilepsy (JME).
    In this paper, they explore to find genes critical to the symptom. First of all physical mapping of 3.5 cM EJM1 was done. Then based on the map, 14 genes in EJM1 from patient family and from healthy family was analyzed. In the process, several YAC clones provided from our division was analyzed by STS-PCR and used for the reference to line up the BAC/PAC contig (assembled).
  • Reference
    Suzuki, T., Delgado-Escueta, A.V., Alonso, M.E., Morita, R., Okamura, N., Sugimoto, Y., Bai, D., Medina, M.T., Bailey, J.N., Rasmussen, A., Ramos-Peek, J., Cordova, S., Rubio-Donnadieu, F., Ochoa, A., Jara-Prado, A., Inazawa, J., Yamakawa, K. Mutation analyses of genes on 6p12-p11 in patients with juvenile myoclonic epilepsy. Neurosci. Lett. 405 (1-2): 126-131, 2006.
  • DNA Resource
    CEPH MEGA YAC clone
    (771C3, 786E1, 834B12, 858B11, 918A11, 938C3, 961C3)

 
Genius method to get rid of the target protein with human culture cells (2016.03.25 K.N.)


A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype. (2016/3/11 T.Y.)

  • Authers had been demonstrated that TRB3 pseudokinase activates cancer cells and also induces nuclear enlargement. To elucidate human-TRB3-induced-mouse-liver-tissue phenotype, They constructed transgenic mice, which has conditional inducible TRB3 by Cre-loxP system. To do so, pCALNL5 (RDB01862) and pxCANCre (RDB01675) provided from our lab were used.
  • Reference
    Sakai, Y., Fukamachi, K., Futakuchi, M., Miyoshi, I., Tsuda, H., Suzui, M., Hayashi, H. A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype. Biol. Pharm. Bull. 37 (6): 1068-1074, 2014.
  • DNA Resource
    pCALNL5 (cat# RDB01862)pxCANCre (cat# RDB01675)


Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation. (2016/2/4 M.O.)

  • Authers had been demonstrated that nomal RNA granules (P-body) formation is perturbed by FUS/TLS mutant protein aggregation induced by its deficient NLS. In this study, they observed P-body dynamycs in living cell cultured various condition using GFP-DCP1 expression vector, which constructed using DCP1 cDNA clone (IRAL010J08) provided from our lab.
  • Reference
    Takanashi, K., Yamaguchi, A., Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation. Biochem. Biophys. Res. Commun. 452 (3): 600-607, 2014.
  • DNA Resource
    IRAL010J08 (cat# HGY084224)

 
Cas9-poly(A) expressing improved plasmid (2016.01.22 T.M.)


Chromatin remodelling and autocrine TNF alpha are required for optimal interleukin-6 expression in activated human neutrophils. (2015/12/18 T.Y.)

  • To elucidate the regulatory mechanism of IL6 expression in human neurophils, authors examined the transcription regulation of upstream region of IL6 gene a part by a luciferase reporter assay. The constructs for this assay were delivated from pGL4-phIL6 RDB07313.
  • Reference
    Zimmermann, M., Aguilera, F.B., Castellucci, M., Rossato, M., Costa, S., Lunardi, C., Ostuni, R., Girolomoni, G., Natoli, G., Bazzoni, F., Tamassia, N., Cassatella, M.A. Chromatin remodelling and autocrine TNF alpha are required for optimal interleukin-6 expression in activated human neutrophils. Nat. Commun. 6: 6061, 2015.
  • DNA Resource
    pGL4-phIL6 (cat# RDB07313)


Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma.(2015/10/23 K.N.)

  • The authors found several genes including LIN28 indicates elevated expression-level in SP cells of the oral squamous cell carcinoma. They also found that the overexpression of LIN28 gene (RDB06602) in the oral squamous cell carcinoma causes aggressive cell activities in terms of proliferation, colony formation and invasion into a solid medium. RDB05956 was used as a control vector.
  • Reference
    Hayashi S, Tanaka J, Okada S, Isobe T, Yamamoto G, Yasuhara R, Irie T, Akiyama C, Kohno Y, Tachikawa T, Mishima K. Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma. Exp. Cell Res. 319 (8) :1220-1228, 2013
  • DNA Resource
    pCMFlag_hsLIN28(cat# RDB06602)
    pCMV_S-FLAG(cat# RDB05956)


Histac for visualization of acetylation of the Histon H4 (2015/9/10 N.N.)

  • The fluorescent probe which detects acetylation of the histone H4 -Histone modification is thought to be one of the mechanisms of epigenetic gene regulation. However, probably because of lack of measures to observe living specimen, spatiotemporal dynamics analysis is in little progress.
  • Here, we offer fluorescent probe “Histac” deposited by Dr. Kazuki Sasaki (RIKEN Brain Science Institute) to detect global hyperacetylation of histone H4 in real-time living cells.
  • Histac consists of an acetylation-binding domain, histone H4 as
    substrate, and the two fluorescent proteins.
  • Under the deacetylated conditions of the histon H4, CFP and Venus in the Histac molecule are closed and yield a yellow fluorescence (emission, 535 nm) by fluorescence resonance energy transfer (FRET). On the other hand, under the acetylated conditions, separation of the fluorescence proteins occurs by the structural change caused by the binding of the bromodomain to histone H4 in the Histac molecule, and yield a blue fluorescence (emission, 480 nm).
  • Observation of the acetylation of histone H4 by the Histaq requires no antibody reaction or fixation of cells. Histac allows successive observation of acetylation of the histone H4 in the introduced living animal cells.
  • In the two original papers, the authors observed fluorescence intensity ratio between CFP versus Venus (480 nm / 535 nm) progressively from 0 to 180 minutes after treatment of histone deacetylase inhibitor, trichostatin A (TSA).
  • The authors proved that Histaq as a powerful tool to show the dynamics of acetylation of histaq H4 in living cells.
  • References
    • Sasaki, K., et al. Real-time imaging of histone H4 hyperacetylation in living cells. PNAS, 106(38):16257-62, 2009.
    • Ito T, et al. Real-Time Imaging of Histone H4K12-Specific Acetylation Determines the Modes of Action of Histone Deacetylase and Bromodomain Inhibitors. Chemistry & Biology, 18(4):495-507, 2011.
    • RIKEN Research. Nov, 2009.
  • DNA Resource
    • Histac pcDNA3.1 (cat# RDB12840)
      For the detection of acetylation at the 5th and 8th lysine residues of histone H4 by the BRDT bromodomain.
    • Histac-K12 pcDNA3.1 (cat# RDB12841)
      For the detection of acetylation at the 12th lysine residues of histone
      H4 by the BRD2 bromodomain.


PRRX1 activates Notch signaling in the invasion of glioblastoma (2015/9/10 N.N.)

  • PRRX1 is involved in the invasion of glioblastoma. It is known that the activation of Notch, Wnt, and Hedgehog pathway also participate in the invasion of the glioblastoma. Sugiyama, M., et al performed a reporter assay using pGa981-6 (as for the Notch signal. cat. #RDB06776), 8×3’Gli-BS-delta51-LucII (as for the Hedgehog signal. cat. #RDB08061) to study activation of the pathways by PRRX1A expression.
  • Reference
    • Sugiyama, M., et al. Paired related homeobox 1 is associated with the invasive properties of glioblastoma cells. Oncol Rep. 33(3):1123-30, 2015.
  • DNA Resource


Vectors for specific expressed in cancer cells. (2015/9/10 T.M.)

  • The HSV-TK gene in pTK5 plasmid was used to construct recombinant adenovirus having AFR promoter for the restricted expression of thymidine kinase in cancer cells.
  • Reference
    Kurayoshi K, et al., Biochem Biophys Res Commun. 450 (1): 240-246 (2014).
  • DNA Resource
    pTK5 (cat# RDB01027)


Involvement of Notch1 inhibition in serum-stimulated glia and oligodendrocyte differentiation from human mesenchymal stem cells (2015/7/31 T.Y.)

  • To elucidate the role of the Notch signaling pathway on human mesenchymal stem cells (KP-hMSCs) to oligodendrocyte differentiation, the KP-hMSCs were transfected with a dominant negative RBP-J (R218H) (cat# RDB03021) to compromise Notch1 function and examined the expression level of differentiation markers. WT-RBP-J plasmid clone (cat# RDB03022) was used as control.
  • Reference
    Lee YJ, Hung SC, Chu MS. Involvement of Notch1 inhibition in serum-stimulated glia and oligodendrocyte differentiation from human mesenchymal stem cells. Stem Cells Cloning 3:165-173, 2010
  • DNA Resource
    pCMX-N/RBP-J (cat# RDB03021)
    pCMX-N/RBP-J (R218H) (cat# RDB03022)


Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation. (2015/7/17 Y.K.)

  • Sir3, a factor of gene silencing in budding yeast contains wH (winged helix-turn-helix domain) domain at C-terminal domain. Authors elucidate a structure and a function of wH domain of Sir3. IRAK013J04 (cat# RDB07566: Human Orc1 clone) was used to subclone its wH domain in E. coli expression vector, and the recombinant protein was compared to Sir3 wH domain.
  • Reference
    Oppikofer M, Kueng S, Keusch JJ, Hassler M, Ladurner AG, Gut H, Gasser SM. Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation. EMBO J. 32 (3): 437-449, 2013
  • DNA Resource
    IRAK013J04 : ORC1L (cat# HGX005420)


WDR81 is necessary for purkinje and photoreceptor cell survival. (2015/7/4 M.O.)

  • A mutant mouse strain nur5 shows tremor, an abnomal gait, Purkinje cell degeneration and photoreceptor cell loss as its phenotype. To indicate that WDR81 gene as the responsible gene for nur5 mouse strain, a BAC clone MSMg01-261K04 was used.
  • Reference
    Traka M, Millen KJ, Collins D, Elbaz B, Kidd GJ, Gomez CM, Popko B. WDR81 is necessary for purkinje and photoreceptor cell survival. J Neurosci. 33 (16): 6834-6844, 2013.
  • DNA Resource
    MSM/Ms Mouse BAC clone
    MSM/Ms mouse BAC references
    MSMg01-261K04 (searching result)


Surface plasmon resonance-biosensor detects the diversity of responses against epidermal growth factor in various carcinoma cell lines. (2015/6/19 T.Y.)

  • Surface plasmon resonance (SPR) – biosensor detects change of angle of resonance (AR) and hence intracellular signaling is monitored. To study the mechanisms of AR change along with activation of EGFR, wild type and mutant EGFR were expressed in cultured cells. To construct expression vectors, pco12 EGFR (cat# RDB01276) was used.
  • Reference
    Hiragun T, Yanase Y, Kose K, Kawaguchi T, Uchida K, Tanaka S, Hide M. Surface plasmon resonance-biosensor detects the diversity of responses against epidermal growth factor in various carcinoma cell lines. Biosens Bioelectron. 32 (1) : 202-207, 2012
  • DNA Resource
    pco12 EGFR (cat# RDB01276)


AID system (2015/3/18 N.N.)

  • To investigate the role of proteins regulated gene expression by chemicals in cells, and gene knock down method is widely applied. Here Dr. Kanemaki Masato offers a new reduction method of interested proteins, Auxin Inducible Degron (AID) system. By addition of auxin, you can degrade proteins in a very short period of time.
  • DNA Resource
    • pNHK60 (cat# RDB08468)
      GFP fused with AID at the C-terminal end is expressed. This clone was used to demonstrate the decay of GFP by addition of auxin (Nishimura, 2009).
    • pMK106 (cat# RDB08469)
      Centromere protein H (CENP-H) fused with the AID at the N-terminal end is expressed.
    • pMK107 (cat# RDB08470)
      CENP-H fused with the AID at the C-terminal end is expressed.
    • Using these resources, the effectiveness of the AID system has been demonstrated (Nishimura, 2009). Remarkably, both N-aid and C-aid cells ceased to grow immediately and subsequently started to die. Compared with the conventional method, it can efficiently remove the target protein.
  • Reference
    • Nishimura, K., et al. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods., 6, 917-922, 2009.
    • Kanemaki, M.T. Frontiers of protein expression control with conditional degrons. Eur. J. Physiol., 465, 419-425, 2013.