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...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2012-02-01
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| Series: | Frontiers in Molecular Neuroscience |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00018/full |
| _version_ | 1831521785729777664 |
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| author | Masahito eYamagata Joshua R Sanes |
| author_facet | Masahito eYamagata Joshua R Sanes |
| author_sort | Masahito eYamagata |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T01:21:03Z |
| format | Article |
| id | doaj.art-00829db5ab184888862f227aba107b93 |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-12-14T01:21:03Z |
| publishDate | 2012-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-00829db5ab184888862f227aba107b932022-12-21T23:22:23ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992012-02-01510.3389/fnmol.2012.0001822331TRANSGENIC STRATEGY FOR IDENTIFYING SYNAPTIC CONNECTIONS IN MICE BY FLUORESCENCE COMPLEMENTATION (GRASP)Masahito eYamagata0Joshua R Sanes1Harvard UniversityHarvard UniversityIn 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.http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00018/fullRetinasynapseGFPneurexinneuroliginphotoreceptor |
| spellingShingle | Masahito eYamagata Joshua R Sanes TRANSGENIC STRATEGY FOR IDENTIFYING SYNAPTIC CONNECTIONS IN MICE BY FLUORESCENCE COMPLEMENTATION (GRASP) Frontiers in Molecular Neuroscience Retina synapse GFP neurexin neuroligin photoreceptor |
| 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 | Retina synapse GFP neurexin neuroligin photoreceptor |
| url | http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00018/full |
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