BRC RESOURCE NEWS
Contents:
- Split-GFP probes visualizing organelle contact sites (2022/12/16)
- StayGold, highly photostable GFP (2022/08/05)
- Mammalian Expression Vector, pEF-BOS series (2022/03/19)
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
(EXR0119e 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. 2022 Apr 25. doi: 10.1038/s41587-022-01278-2. Epub ahead of print. 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
- RDB19605 (n1)StayGold/pRSET
(EXR0118e 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
(EXR0117e 2022.03.19 N.N)
(GRP0071e 2022.03.19 N.N)
2022.12.16
