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dnaconda01

 

 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
    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.

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.

 

  • 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.

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.

 

  • 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

 

  • 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

 

  • 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

 

  • 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.

 

  • 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

 

  • Vectors for specific expressed in cancer cells.
    • 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).