NIR-GECO1 provides a robust inverted fluorescence response (i.e., a fluorescence decrease upon Ca 2+ increase) in response to Ca 2+ concentration changes in cultured cells, primary neurons, and acute brain slices. NIR-GECO1 was engineered by genetic insertion of the Ca 2+-responsive domain calmodulin (CaM)-RS20 into the protein loop close to the BV binding site of mIFP. We recently described the conversion of an NIR FP (mIFP, a biliverdin (BV)-binding NIR FP engineered from the PAS and GAF domains of Bradyrhizobium bacteriophytochrome) into a GECI designated NIR-GECO1. In contrast, efforts to develop GECIs with near-infrared (NIR) excitation and emission (>650 nm) are at a relatively nascent state.
SNAPGENE CODON OPTIMIZATION SERIES
These efforts have produced a series of high-performance GECIs that are highly optimized in terms of brightness, kinetics, Ca 2+ affinities, cooperativity, and resting (baseline) fluorescence. Over a time frame spanning 2 decades, tremendous effort has been invested in the development of visible wavelength GECIs based on green and red fluorescent proteins (GFP and RFP, respectively). Induced pluripotent stem cell-derived cardiomyocyte IUE,įluorescence imaging of intracellular calcium ion (Ca 2+) transients using genetically encoded Ca 2+ indicators (GECIs) is a powerful and effective technique to monitor in vivo neuron activity in model organisms. Genetically encoded calcium ion indicator GFP, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors declare no competing interests. Harrington Fellowship from the faculty of medicine at the University of McGill. acknowledges the Foundation of Westlake University. was supported by a grant from NSERC (RGPIN-2017-05005). was supported by grants from CIHR (FDN-143238) and Brain Canada. acknowledges Lisa Yang, John Doerr, the HHMI-Simons Faculty Scholars Program, the HHMI Investigator Program, U.S.-Israel Binational Science Foundation (2014509), NIH (2R01-DA029639, 1R01-MH12297101, 1R01-DA045549, and 1RF1-NS113287), Human Frontier Science Program (RGP0015/2016), and NSF (18480870). 159605), and pSF11-wNIR-GECO2-T2A-HO1(plasmid no.159606) are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement.įunding: Work in the lab of R.E.C. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting information. Received: ApAccepted: OctoPublished: November 24, 2020Ĭopyright: © 2020 Qian et al. Lishko, University of California, Berkeley, UNITED STATES PLoS Biol 18(11):Īcademic Editor: Polina V. (2020) Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca 2+ dynamics.Ĭitation: Qian Y, Cosio DMO, Piatkevich KD, Aufmkolk S, Su W-C, Celiker OT, et al. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Near-infrared (NIR) genetically encoded calcium ion (Ca 2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues.
0 kommentar(er)
