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      Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems

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          Abstract

          Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1×10 11 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1×10 12 vg AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust co-transduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell type-specific promoters, these AAVs provide targeted gene expression across the nervous system and enable efficient and versatile gene manipulation throughout the nervous system of transgenic and non-transgenic animals.

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          Most cited references43

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          A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

          Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.
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            Single-cell phenotyping within transparent intact tissue through whole-body clearing.

            Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies. Copyright © 2014 Elsevier Inc. All rights reserved.
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              Improving FRET dynamic range with bright green and red fluorescent proteins

              A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.
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                Author and article information

                Journal
                9809671
                21092
                Nat Neurosci
                Nat. Neurosci.
                Nature neuroscience
                1097-6256
                1546-1726
                27 May 2017
                26 June 2017
                August 2017
                26 December 2017
                : 20
                : 8
                : 1172-1179
                Affiliations
                Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
                Author notes
                [* ]To whom correspondence should be addressed: Viviana Gradinaru, Ph.D., Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Blvd. MC 156-29, Pasadena, CA 91125, Phone: (626) 395-6813, viviana@ 123456caltech.edu , Benjamin E. Deverman, Ph.D., Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Blvd. MC 139-74, Pasadena, CA 91125, Phone: (626) 395-2776, bd@ 123456caltech.edu
                Article
                NIHMS879643
                10.1038/nn.4593
                5529245
                28671695
                f9ae0e32-5161-484d-a9f0-ff3211a84c71

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                Neurosciences
                Neurosciences

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