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BRC RESOURCE NEWS

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A non-viral gene transfection vector, piggyBac®

It is an expression system that allows easy integration and excision of transposons into the genome, so please use it when establishing stable cell lines. DNA Bank provides pB vectors that match research needs, such as those capable of expressing hCas3 for CRISPR/Cas systems (RDB18213 pPB-CAG-hCas3).

(INF0090e 2025.02.17 N.N.)

A bicistronic cell cycle reporter, Fucci2a

  • Fucci2a has G1 phase specific protein (Cdt1(30/120) -mCherry) and S/G2/M phase specific protein (Geminin(1/110) -mVenus) tethered by a T2A sequence. Because the expression levels of the two fluorescent proteins are steady when introduced into cultured cells or when using the Tg mouse model, the cell cycle can be observed with high accuracy.

(INF0089e 2024.09.13 N.N.)

Common marmoset cDNA Clone MMTD

(INF0088e 2024.06.03 N.N.)

Monomeric StayGold, highly photostable GFP

  • Please refer to the following for the plasmid list.
    Ando, R. et al., StayGold variants for molecular fusion and membrane-targeting applications. Nat Methods. 2023 Nov 30. doi: 10.1038/s41592-023-02085-6. Epub ahead of print PMID 38036853.

(INF0087e 2024.02.13 N.N.)

Various fluorescent proteins such as photoswitchable fluorescent protein, Kohinoor

  • Dr. Takeharu Nagai, Osaka university Insitute of Scientic and Industrial Reserch Nagai-laboratory has deposited fluorescent proteins such as photoswitchable FP Kohinoor, acid resistant monomeric GFP Gamillus and short-wavelength FP Sirius.
    • Fluorescent Protein Product PMID Resource name Cat ♯
    Kohinoor 2.0 A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging 33481018 Kohinoor 2.0/pcDNA3 RDB19862
    SPOON Spontaneously blinking luorescent protein for simple single laser super-resolution live cell imaginga 29963852 SPOON/pcDNA3 RDB19863
    Gamillus Acid-Tolerant Monomeric GFP from Olindias formosa 29290624 Gamillus/pcDNA3 RDB19864
    Super Nova Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation 29712573 SuperNova Green/ pRSETB RDB19865
    Sirius A monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation 19349978 Sirius/pcDNA3 RDB19875
    Cameleon-Nano An ultrasensitive calcium ion sensor with Kd, 65 nM
    (Also available in 15, 30 and 50 nM versions)    		 2069399 Yellow Cameleon-Nano65 /pcDNA3 RDB19877

(INF0086e 2023.09.01 N.N.)

High intensity forms of luciferase and luminescent proteins from various organisms (2023/04/28)

  • In nature, there are many bioluminescent organisms such as fireflies and a part of marine life. The bioluminescence is a result of oxidation of substrates. Wide variety of research methods applying bioluminescence, such as reporter assays, have been developed and used widely.
  • Of reporter proteins, luciferase from the Photinus pyralis is a well-known luminescent enzyme, but in recent years, new luminescent enzymes have been derived from various marine organisms such as copepods and shrimp and artificially designed luminescent enzymes also have been developed. They have smaller molecular weight and higher intensity than that from fireflies. These characteristics less affect the function of proteins of interest fused to them, and the luminescent proteins are applied to imaging research observing the real-time dynamics of the target proteins in living cells.
  • JNC CORPORATION (JNC), which sold bioluminescent plasmids developed in the course of bioluminescence research, deposited 27 plasmid DNAs carrying luminescent protein genes such as from Gaussia princeps, Oplophorus gracilirostris and Aquorin from Aequorea victoria. We will take over them and start to provide.
    URL：JNC CORPORATION’s Resource
  • Dr. KIM Sung Bae and his colleagues at the National Institute of Advanced Industrial Science and Technology (AIST, Japan) have developed Artificial luciferase (ALuc), which is based on a luminescent enzymes derived from copepods and artificially reconstituted with optimal amino acid sequences. ALuc shows 100 times higher intensity and prolonged luminescence than original enzymes (Kim, S.B. et al. Bioconjug. Chem., 2013). They have deposited two types of ALuc plasmids to us this time.
    URL：Sung-Bae Kim’s Resource
  • Other available products include red-shifted luciferase (Akaluc) and luciferase that is less susceptible to environmental factors (PmatLuc1, PsagRE1Luc, etc.). Choose according to your research application. We look forward to receiving your orders!
    URL：Higher intensity luciferase
  • Reference
    • Inouye, S. et al. Proc. Natl. Acad. Sci. U S A. 82 (10): 3154-3158, 1985. PMID 3858813
    • Inouye, S. et al. Protein Expr. Purif. 109: 47-54, 2015. PMID 25665506
    • Inouye, S. et al. Protein Expr. Purif. 52: 66-73, 2007. PMID 16997571
    • Inouye, S. FEBS Lett. 347 (2-3): 163-168, 1994. PMID 8033996
    • Inouye, S. et al. FEBS Lett. 315 (3): 343-346, 1993. PMID 8422928
    • Inouye, S. J. Biochem. 143 (5):711-717, 2008. PMID 18296715
    • Inouye, S. et al. FEBS Lett. 341 (2-3): 277-280, 1994. PMID 8137953
    • Inouye, S. et al. FEBS Lett. 481 (1): 19-25, 2000. PMID 10984608
    • Inouye, S. et al. Biochem. Biophys. Res. Commun. 445 (1): 157-162, 2014. PMID 24491536
    • Inouye, S. et al. Biochem. Biosci. Biotechnol. 75 (3): 568-571, 2011. PMID 21389603
    • Inouye, S. et al. Biochem. Biophys. Res. Commun. 376 (3): 448-453, 2008. PMID 18789309
    • Suzuki, T. et al. Anal. Bichem. 415 (2): 182-189, 2011. PMID 21477579
    • Suzuki, T. et al. Plos One. 6 (9): e25243, 2011. PMID 21969874
    • Inouye, S. et al. Protein Expr. Purif. 128: 93-100, 2016. PMID 27506135
    • Inouye, S. & Sahara, Y. Protein Expr. Purif. 53 (2): 384-389, 2007. PMID 17275329
    • Kim, S.B. et al. Bioconjug. Chem. 24 (12): 2067-2075, 2013. PMID 24237362
    • Kim, S.B. et al. ACS Comb. Sci. 19 (9): 594-599, 2017. PMID 28742969

(INF0085e 2023.04.28 N.N.)

Split-GFP probes visualizing organelle contact sites (2022/12/16)

  • Most observations of organelle contact sites are usually performed using fixed cells. Methods of analysis in living cells have also been reported, but there have been problems such as low sensitivity. A research group led by Dr. Yasushi Tamura of Yamagata University and Dr. Toshiya Endo of Kyoto Sangyo University has developed new research tool to visualize organelle contact sites by taking advantage of the unique characteristics of Split-GFP (Kakimoto, Y. et al., Sci. Rep. 8 (1): 6175, 2018. PMID 29670150).
  • Split-GFP is a set of protein fragments of GFP that is composed of two parts, each of which lose their fluorescence. When the split-GFP fragments come into contact, the mature GFP protein is reconstituted and restores its fluorescence. Of 11 beta strands, the research group expressed the 1st to 10th (GFP1-10) and the 11th (GFP11) strands of GFP fused to distinct proteins localizing on different organelles. Since the fluorescence derived from reconstituted Split-GFP was detected corresponding to the shape of organelle contact sites and locations of known organelle binding factors, it was confirmed that the organelle contact sites was actually visualized in living cells.
  • Dr. Tamura kindly deposited split-GFP probe plasmids which express GFP1-10 and GFP11 fused with localization sequences to mitochondria, endoplasmic reticulum, etc., for mammalian cell expression and for genome integration of S. cerevisiae. We look forward to receiving your orders!
  • List of plasmids

  • Reference
    • Kakimoto, Y. et al., Sci. Rep. 8 (1): 6175, 2018. PMID 29670150
    • Tashiro, S. et al., Front. Cell Dev. Biol., 8: 571388, 2020. PMID 33330450
    • Shirane, M. et al., Nat. Commun., 11 (1): 4576, 2020. PMID 32917905

(INF0084e 2022.12.16 N.N.)

StayGold, highly photostable GFP (2022/08/05)

  • Since the discovery of green fluorescent protein (GFP) from Aequorea victoria, many fluorescent proteins with various wavelength ranges have been developed and used as visualization tools for analyzing the localization and dynamics of target genes in cells. However, fluorescent proteins have the weakness that they fade under strong or repeated excitation light exposure.
  • A collaborative research group led by Dr. Atsushi Miyawaki of RIKEN Neuroscience Research Center and Tohoku University, Kitasato University, and Kao Corporation has isolated a novel GFP, CU17S gene derived from the jellyfish Cytaeis uchidae. They introduced the CU17S gene into cultured cells and E. coli and found that it was highly photostable. Furthermore, they succeeded in substantially increasing the brightness of the fluorescent protein without compromising its photostability by introducing a specific mutation in the CU17S gene, and this fluorescent protein was called “StayGold” （Hirano, M. et al., Nat. Biotechnol. 2022. PMID: 35468954）.
  • The photostability of StayGold is up to hundredfold better than existing fluorescent proteins. Taking advantage of the photostability of StayGold, Dr. Atsushi Miyawaki and his colleagues conducted time-dependent observations to reveal structural changes in intracellular organs. In addition, by combining StayGold with VHH antibody to spike protein of SARS-CoV-2, they succeeded in observing the maturation process of virus particles in infected cells.
  • Dr. Atsushi Miyawaki has deposited a total of 10 plasmids, including StayGold (cat# RDB19605: (n1)StayGold/pRSET, cat# RDB19606: (n1)StayGold(c4)/pRSET), which can fuse target genes to the N-terminus or C-terminus, and tandem dimer StayGold (cat# RDB19609: tdStayGold/pcDNA3), which can fluorescently label microtubule gliding molecules. We look forward to receiving your orders!

  • Please refer to the following for the plasmid list.
    Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40 (7): 1132-1142 (2022). PMID 35468954.

    • RDB19605  (n1)StayGold/pRSET
      Fluorescent protein StayGold for fusion to the C-terminus of target molecule
    • RDB19606  (n1)StayGold(c4)/pRSET
      Fluorescent protein StayGold for fusion to the N-terminus of target molecule
    • RDB19607  (n1)oxStayGold/pRSET
      Fluorescent protein oxStayGold for fusion to the C-terminus of target molecule
    • RDB19608  (n1)oxStayGold(c4)/pRSET
      Fluorescent protein oxStayGold for fusion to the N-terminus of target molecule
    • RDB19609  tdStayGold/pcDNA3
      Fluorescent protein tdStayGold for fusion to the C-terminus of target molecule
    • RDB19610  tdStayGold(c4)/pBS Coupler
      Fluorescent protein tdStayGold for fusion to the N-terminus of target molecule
    • RDB19611  tdoxStayGold/pcDNA3
      Fluorescent protein tdoxStayGold for fusion to the C-terminus of target molecule
    • RDB19612  tdoxStayGold(c4)/pBS Coupler
      Fluorescent protein tdoxStayGold for fusion to the N-terminus of target molecule
    • RDB19613  er-(n2)oxStayGold(c4)
      Vector for ER labeling with fluorescent protein oxStayGold
    • RDB19614  mt-(n1)StayGold
      Vector for Mitochondria labeling with fluorescent protein StayGold

(INF0083e 2022.08.05 N.N.)

Mammalian Expression Vector, pEF-BOS series (2022/03/19)

  • pEF-BOS and pEF-BOS-EX are mammalian expression vectors developed by Dr. Nagata Shigekazu of Immunology Frontier Research Center, Osaka University (Mizushima, S and Nagata, S. Nucleic Acids Res. 18 (17): 5322, 1990. Murai, K. et al. Proc. Natl. Acad. Sci. USA. 95 (7): 3461-3466, 1998). These vectors carry the human elongation factor one alpha promoter (EF-1 alpha) with high promoter activity independent of cell type and are used in gene transfer experiments into various cell types.
  • We will now provide the new modified pEF-BOS-EX vectors deposited by Dr. Nagata. pEF-Flag-EX (RDB18972) is utilized to express gene products with a FLAG-tag. pNEF-BOS-EX (RDB18970) and pPEF-BOS-EX (RDB18971) with neomycin and puromycin resistance markers, respectively, are utilized to establish stable expression cell lines. We are looking forward to receiving your request for pEF-BOS vector series.
    Cat.# Resource name Remarks
    RDB07939 pEF-BOS  
    RDB07940 pEF-BOS-EX  
    RDB18972 pEF-Flag-EX N-terminal FLAG-tag
    RDB18970 pNEF-BOS-EX Selectable markers in mammalian cells, Neomycin
    RDB18971 pPEF-BOS-EX Selectable markers in mammalian cells, Puromycin

     

(INF0082e 2022.03.19 N.N.)

(GRP0071e First Publication Date 2022.03.19 N.N.)

2024.09.13



 

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