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News
Information to Residents of the European Economic Area (EEA)
News
Seeking a Principle Investigator (indefinite-term position), Gene Engineering Division (RIKEN BRC Gene Bank) (2019/07/26 T.M.)
New Director Appointed (2019/04/01 T.M.)
Congratulations to Distinguished Professor Tasuku Honjo for the 2018 Nobel Prize! (2018/10/3 T.M.)
- The 2018 Nobel Prize in Physiology or Medicine for the discovery of cancer therapy by inhibition of negative immune regulation
Genetic resources regarding the prize are available (2018/10/3 T.M.)
Dnaconda’s Recommendation
 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.
[more…]
- 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.
[more…]
- 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)
Expression vector of optogenetics technique (2019/1/11 N.N.)
- New plasmids developed by Dr. Toru Ishizuka of Tohoku University are available.[more…]
- 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)
mCherry expression vector with Cre/loxP system (2018/12/07 S.H.)
- Dr. Yumiko Hatanaka and her colleagues at Osaka University developed mCherry expression vector with Cre/loxP system.[more…]
- 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 (2018/11/13 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. [more]
- 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
Previous articles..
Resource Information
 Function of transcription coactivator TAZ on the regulation of IL1B expression. (2019/5/13 T.M.)
- Involvement of YAP on the phenotype of malignant mesothelioma (MM) has been demonstrated. However, the function of TAZ, a paralog of YAP, in MM cells has not been clarified. [more…]
- Here the authors demonstrated that an activated form of TAZ (S89A) can transform immortalized mesothelial cells. By microarray analysis of mRNA expression and a gene ontology analysis suggested that TAZ might be involved in the cytokine gene regulation pathway. The function of TAZ on the malignant phenotype were tested by mRNA expression of IL1A, IL1B, IL6 and IL24 after TAZ activation and effect of knock down of them on the proliferation of cells. Of those, IL1B was found as downstream gene of TAZ activation.
- To test whether TAZ activate transcription of IL1B, pKM2L-phIL1B luciferase reporter was utilized.
- Reference
Matsushita, A., Sato, T., Mukai, S., Fujishita, T., Mishiro-Sato, E., Okuda, M., Aoki, M., Hasegawa, Y., Sekido, Y. TAZ activation by Hippo pathway dysregulation induces cytokine gene expression and promotes mesothelial cell transformation. Oncogene 38: 1966-1978 (2019). PMID 30401981.
- Plssmid clone
pKM2L-phIL1B (cat# RDB05526)
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. [more…]
- 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)
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-LC3ΔG (lacking glycine (G) at the LC 3 end) simultaneously at equimolar amounts.[more…]
- As autophagy progresses, the fluorescence of GFP-LC3 decays whereas that of RFP-LC3ΔG 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)
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. [more]
Stable ChIP-seq analysis using transcription factors tagged with biotinylated peptide tag. (2018/2/8 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.[more]
- 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.[more]
- 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]
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