Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572

Nature Biotechnology (Impact Factor: 41.51). 01/2005; 22(12):1567-72. DOI: 10.1038/nbt1037
Source: PubMed


Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

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Available from: Nathan C Shaner, Jan 21, 2015
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    • "Generation of GalR1-mCherry-[neo + ] and GalR2-hrGFP-[neo + ] knockin mice Mouse genomic clones including either GalR1 or GalR2 genes from the bMQ mouse strain 129S7 (129Sv) bacterial artificial chromosome (BAC) library (inserts 89–178 kb; Source BioScience; Adams et al., 2005) were electroporated into strain EL250 Escherichia coli (Lee et al., 2001). This allowed temperature-inducible, lambda Red-mediated, homologous recombination into the BAC (Copeland et al., 2001; Lee et al., 2001) of PCR products from either vector hrGFP-FRTneoFRT (Balthasar et al., 2004; Parton et al., 2007; hrGFP derived from Stratagene-Agilent vector phrGFP-1, Zeng et al., 2003; Fig. 2A, middle panel) flanked by GalR2 homologous sequence or vector mCherry- FRTneoFRT (hrGFP exchanged for mCherry; Shaner et al., 2004) flanked by GalR1 homologous sequence. Within the latter PCR product an AseI restriction site was introduced immediately downstream of the 3′ FRT site (Fig. 1A, middle panel), for use in Southern blot digests, and the mCherry/hrGFP heterologous 3′-untranslated region (UTR) has identity to nt 705–1193 of vector pCMV-Script (AF028239) which includes the SV40 early region poly(A) site (Connelly and Manley, 1988; J02400, nt 2828–2547). "
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    ABSTRACT: The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1 and Gal2 and the less studied Gal3 (GalR1-3 gene products). There is a wealth of data on expression of Gal1-3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). Copyright © 2015. Published by Elsevier Inc.
    Molecular and Cellular Neuroscience 08/2015; 68. DOI:10.1016/j.mcn.2015.08.006 · 3.84 Impact Factor
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    • "Nonetheless, FPs have shown great promise for in vivo optoacoustic imaging in zebrafish [9] [20], glioma visualization [8], multi-contrast optoacoustic imaging [21] and optoacoustic flow cytometry [22]. Many fluorescent proteins lack photostability [16] [18], which may impede quantitative measurements [23]. To this end, nanosecond-laser-induced photobleaching has been shown to occur in isolated FPs [24]. "
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    ABSTRACT: Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 08/2015; 69:38-44. DOI:10.1016/j.biomaterials.2015.07.051 · 8.56 Impact Factor
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    • "The mitochondrial localization of RRL–GFP was further confirmed by co-localization with a mitochondrion-selective dye MitoTracker Red (Fig. 1A–C). Additionally, the mitochondrial localization of RRL–GFP was also determined by tobacco leaf infiltration experiments with a well-known mitochondria marker, mt-rk CD3-991 (Nelson et al., 2007), which was generated with a red fluorescent protein, mCherry (Shaner et al., 2004). The RRL–GFP fusion protein co-localized with mt-rk CD3-991 by fluorescence analyses (Fig. 1D–F). "
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