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Oncogenic effects of evolutionarily conserved noncoding RNAs “ECONEXIN” on glioma formation (2020/11/10 K.N.)
- It has been shown that long noncording RNAs (lncRNAs) play significant roles on oncogenic transformation. However, the function of most lncRNAs is uncharacterized and it is very tough to find new candidates of cancer-associated lncRNAs. [more…]
- Based on the hypothesis that the sequences of genes with important functions are highly conserved during evolution, authors identified human lncRNA gene “ECONEXIN” (LINC00461) and the mouse C130071C03RIK gene that is highly homologous to humans. They showed that ECONEXIN is highly expressed in brain, especially in glioma, interacts to Argonaute protein (AGO2), which has a function of RNA mediated silencing, together with miR-411-5p. They also figured out that ECONEXIN-[miR-411-5p]-AGO2 complex repress the expression of Topoisomerase2A (TOP2A), which downregulates gliomagenesis.
- Vectors constructed in this study, AGO2 wt and PAZ domain mutants expression vectors (RDB15797-15799), a luciferase assay vector for TOP2A-3’UTR (RDB16800), and TOP2A expression vector (RDB16801) were deposited in RIKEN BRC.
- Reference
Deguchi, S. et al. Oncogenic effects of evolutionarily conserved noncoding RNA ECONEXIN on gliomagenesis. Oncogene 36 (32): 4629-4640, 2017. PMID: 28368417.
- DNA Resource
p3xFLAG-Myc-CMV-AGO2-WT (cat# RDB15797)
p3xFLAG-Myc-CMV-AGO2-PAZ-mut (cat# RDB15798)
p3xFLAG-Myc-CMV-AGO2-PAZ-del (cat# RDB15799)
pmirGLO-TOP2A-3′-UTR (cat# RDB15800)
pcDNA-DEST47-TOP2A (cat# RDB15801)
The substrate specificity of deubiquitinase “USP -25” that recognizes the polyubiquitin chain linked to Lys 48 is determined by the tandem ubiquitin interacting motif (UIM) region (2020/11/10 K.N.)
- Ubiquitin not only modifies other proteins to promote their degradation in proteosomes, but also plays various roles. There are two different structure of polyubiquitin chain, “Lys48-link” and “Lys63-link”, based on the binding residue. [more…]
- It has not been well-documented the detail mechanism of deubiquitination of Lys48-link polyubiquitin than that of Lys63-link polyubiquitin chain. Authors constructed the expression vectors of ubiquitin binding domain mutants of deubiquitinase USP25 and elucidated a important role of tandem ubiquitin interacting motif (UIM) on the enzymatic activity as well as ubstrate selectivity of Lys48-link polyubiquitin. The wild-type and mutants of USP25 cDNA was constructed from the USP25 cDNA of the human cDNA clones of the Genome Network Project available form RIKEN BRC.
- 論文
Kawaguchi, K. et al. Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25. Sci. Rep. 7: 45037, 2017. PMID 28327663.
- DNA Resource
IRAK168O08 (cat# HGY067544)
Development of a method using CRISPR-Cas9 to delete the desired large genomic region (2020/11/10 K.N.)
- Genome-editing technology using CRISPR-Cas9 has made it possible to establish mice lacking the desired genome, for example exons. However, it is still difficult to eliminate regions that exceed the megabases and have the desired break points. [more…]
- Such method must be overcome to establish a model mouse that mimics human disease involving domain defects of several megabase units. Authors show an example in which a 5 megabase deletion with the desired breakpoint was created by genome editing of a mouse fertilized egg using a donor vector with a designed breakpoint upstream of the mouse Sox9 gene. “B6N mouse BAC clone” located upstream of mouse Sox9 were used for FISH probe to identify breakpoints.
- Reference
Kato, T. et al. Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system. Sci. Rep. 7 (1): 59, 2017. PMID: 28246396.
- DNA resources
B6N mouse BAC clones
B6Ng01-332K06, B6Ng01-295D01、 B6Ng01-223B06, B6Ng01-111E12, B6Ng01-299I16, B6Ng01-234K06
Long noncoding RNAs “TUG1” regulated by Notch signals are targets for glioma therapy. (2020/11/10 K.N.)
- Notch signaling promotes self-renewal of glioma stem cells (GSC). The detailed mechanism through the activity of SOX2, MYC and NESTIN etc. is not clearly understood. Screened a long noncoding RNA by genome informatics analysis and expression analysis, the authors identified “TUG1 (taurine upregulated gene 1)” as one of the genes expressing on Notch signal dependent manner. [more…]
- The authors showed that TUG1 works as an antagonist of miR-145 which suppresses function of important factors for GSCs self-renewal such as SOX2 and MYC, that TUG1 indirectly downregulates neuronal growth factors such as BDNF via methylation of H3K27 and that the reduction of TUG1 expression resulted in the suppression of GSC growth. Furthermore, they found exon 1 of the TUG1 has a functional domain by using expression vectors of each exon of the TUG1. Those vectors were available from RIKEN BRC.
- Reference
Katsushima, K., Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment. Nat. Commun. 7: 13616 (2016). PMID 27922002.
- DNA Resource
pTnT-TUG1 exon1 (RDB15794)
pTnT-TUG1 exon2 (RDB15795)
pTnT-TUG1 exon3 (RDB15796)
Achilles, a novel fluorescent protein.(2020/3/24 N.N.)
- For live-cell imaging using fluorescent proteins, it is important to use a fluorescent protein having short maturation time. Dr. Yusuke Niino and Dr. Atsushi Miyawaki of RIKEN Center for Brain Science (CBS) have developed Achilles which has shorter maturation time than Venus. Achilles is available now from DNA Bank. [more…]
- Dr. Ryoichiro Kageyama of Kyoto University and his colleagues tried to observe the dynamics of the segmentation clock Hes7 using a Venus-Hes7 fusion protein, but it was hard to detect Venus fluorescence due to the short half-life of Hes7 protein. However, they employed the newly developed Achilles instead of Venus and succeeded analyses of the dynamics of the Hes7 protein (Yoshioka-Kobayashi, K. et al. Nature, 2020).
- Periodic transcriptional changes of the segmental clock gene Hes7 can be reproduced in cultured cells. Dr. Olivier Pourquie of Harvard Medical School and his colleagues established a cell line in which destabilized Achilles with a proteolytic signal was inserted into the 3 ‘end of the Hes7 gene. They were able to detect oscillatory expression of the Hes7-Achilles gene at the single-cell level. (Diaz-Cuadros, M. et al. Nature, 2020).
- Reference
Yoshioka-Kobayashi, K. et al. Coupling delay controls synchronized oscillation in the segmentation clock. Nature, 2020. PMID: 31915376
Diaz-Cuadros, M. et al. In vitro characterization of the human segmentation clock. Nature, 2020. PMID: 31915384
- DNA Resource
Achilles_pRSETB (cat# RDB15982)
Glucocorticoid up-regulates PD-1 on T cells.(2020/2/18 N.N.)
- T cell activation is known to be suppressed by an immunosuppressant Glucocorticoid (GC) and inductively expressed PD-1. [more…]
- Maeda, N. et al. found the enhancement of PD-1 expression by GC during their study of the effect of immunosuppressants on PD-1 expression. Furthermore, they found that the up-regulation of PD-1 was caused by the transcriptional activation of PD-1 via the GC response element on the transcription regulatory region located upstream of the PD-1 gene. The genomic fragment of the transcriptional regulatory region of PD-1 used for this analysis was amplified by PCR from the B6N BAC clone B6Ng01-240G08 provided by us.
- Reference, Related article
Maeda, N. et al. Glucocorticoids potentiate the inhibitory capacity of programmed cell death 1 by up-regulating its expression on T cells. J. Biol. Chem., 294 (52): 19896-19906, 2019. PMID: 31723031
- Resource information
Clone Set, Library & Genomic -Genomic clone-
Novel vectors for efficient PCR cloning (2019/10/11 N.N.)
- Novel vectors for efficient cloning of PCR products by TA-cloning, blunt-end cloning and both cloning methods were recently developed and published in April of this year as described below, by Dr. Ken Motohashi of the Kyoto Sangyo University and are now available from us. We are looking forward to hearing your order![more…]
- Deposited resources
- TA cloning vector; pCRT (cat# RDB17479)
By digestion with Xcm I restriction enzyme, it can be used for TA cloning.
- Blunt-end cloning vector; pCRZero (cat# RDB17481)
By digestion with EcoR V restriction enzyme, it can be used for blunt-end cloning.
- TA- and Blunt-end cloning vector; pCRZeroT (cat# RDB17480)
This vector can be used for the TA cloning or blunt-end cloning by digestion with either Xcm I or Sma I, respectively.
- Reference
Motohashi, K. A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products. Sci. Rep. 9 (1): 6417, 2019. PMID: 31015513.
Elucidation of control system for regulating the morphology and orientation of cells in animal tissues. (2019/9/6 N.N.)
- The morphology and orientation of cells in tissues are determined by structural dynamics of the cytoskeleton and polarity of cells. It has been suggested that the structural dynamics of the cytoskeleton is induced during the exchange of signal transduction molecules. However, controlling the structural dynamics by the molecules remained to be elucidated.
[more…]
- Dr. Koji Kikuchi and his colleagues of the University of Kumamoto proposed a regulatory mechanism that links Wnt5a signaling pathway and microtubule dynamics.
- Based on the fact that the Wnt5a signaling pathway is involved in the regulation of the microtubules that make up the cytoskeleton, they identified Map7/7D1 after phenotypic screening to search intervening molecules using an siRNA library. Subsequently, they revealed that Map7/7D1 binds to Disheveled (Dvl), which is one of the mediators in the Wnt5a signal pathway, and controls the localization of Dvl as results of experiments including knock down of Map7/7D1 in cultured cells and Drosophila mutants.
- In this study, they constructed pEGFP-N3-hMap7 to study intracellular localization of Map7/7D1 by means of the fluorescence live imaging. This clone contains human MAP7 cDNA derived from IRAK049L15 provided by us. Three clones including pEGFP-N3-hMap7 were deposited by Dr. Koji Kikuchi. We are looking forward to receiving your request.
- Reference
Kikuchi, K. et al. Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling. EMBO Rep. 19 (7): e45471, 2018. PMID: 29880710.
- Deposited and Provided resources
pEGFP-N3-hMap7 (cat# RDB16943)
pcDNA3.1-V5His6-hMap7 (cat# RDB16944)
pEGFP-N3-mMap7D1 (cat# RDB16945)
IRAK049L15 (cat# HGX019879)
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Resource Information
Distribution of BAC clones (2020/2/18 N.N.)
- RIKEN BRC DNA Bank distributes the following Bacterial Artificial Chromosomes (BAC) clones; Mouse (C57BL/6N and MSM/Ms strains), Rat (F344/Stm and LE/Stm strains), Japanese macaque (M. fuscacta), and Drosophila flies (5 species, including D. melanogaster).[more…]
- These BAC clones can be searched by a gene symbols as a key word, and the position of the clone on the genome region can be visually confirmed.
- BAC clones are used to construct gene knock out (KO) vectors and reporter genes having fluorescent proteins and LacZ to establish KO and transgenic animals. The BAC clones are also used for cloning the transcriptional regulatory region of genes.
- One of successful use of BAC clone is described in “Glucocorticoid up-regulates PD-1 on T cells (2020/2/18 N.N.)”.
- Resource information
Clone Set, Library & Genomic -Genomic clone-
Atg2 mediates direct lipid transfer between membranes for autophagosome formation. (2019/12/16 N.K.)
- Autophagy is one of the intracellular recycling systems of protein. Unrequired proteins to be degraded are first isolated in the organelle “autophagosome” formed by phospholipid membrane. [more…]
- The autophagosome biogenesis are catalyzed by several Atg proteins. In this study, authors analyzed X-ray crystallography and lipid transfer activity of Atg2 protein. They elucidated that Atg2 have tethering activity between ER membrane and autophagosome membrane and also have phospholipid transfer activity between them. Recombinant Atg2 protein of budding yeast and Atg2 protein of fisson yeast were used in this study. To construct expression vector of Atg2 (fission yeast), SpFFH31A11(SPW052411) provided from Riken BRC were used.
- Reference
Osawa, T. et al. Atg2 mediates direct lipid transfer between membranes for autophagosome formation. PMID 30911189.
- Plssmid clone
SpFFH31A11 (SPW052411)
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]
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