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Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP)
In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2012
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280602/ https://www.ncbi.nlm.nih.gov/pubmed/22355283 http://dx.doi.org/10.3389/fnmol.2012.00018 |
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author | Yamagata, Masahito Sanes, Joshua R. |
author_facet | Yamagata, Masahito Sanes, Joshua R. |
author_sort | Yamagata, Masahito |
collection | PubMed |
description | In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method. |
format | Online Article Text |
id | pubmed-3280602 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-32806022012-02-21 Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) Yamagata, Masahito Sanes, Joshua R. Front Mol Neurosci Neuroscience In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method. Frontiers Media S.A. 2012-02-16 /pmc/articles/PMC3280602/ /pubmed/22355283 http://dx.doi.org/10.3389/fnmol.2012.00018 Text en Copyright © 2012 Yamagata and Sanes. http://www.frontiersin.org/licenseagreement This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited. |
spellingShingle | Neuroscience Yamagata, Masahito Sanes, Joshua R. Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title_full | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title_fullStr | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title_full_unstemmed | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title_short | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP) |
title_sort | transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (grasp) |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280602/ https://www.ncbi.nlm.nih.gov/pubmed/22355283 http://dx.doi.org/10.3389/fnmol.2012.00018 |
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