Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP
Summary: Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2017-04-01
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| Series: | Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124717303947 |
| _version_ | 1818457528828166144 |
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| author | Alexander R. Nectow Maria V. Moya Mats I. Ekstrand Awni Mousa Kelly L. McGuire Caroline E. Sferrazza Bianca C. Field Gabrielle S. Rabinowitz Kirsty Sawicka Yupu Liang Jeffrey M. Friedman Nathaniel Heintz Eric F. Schmidt |
| author_facet | Alexander R. Nectow Maria V. Moya Mats I. Ekstrand Awni Mousa Kelly L. McGuire Caroline E. Sferrazza Bianca C. Field Gabrielle S. Rabinowitz Kirsty Sawicka Yupu Liang Jeffrey M. Friedman Nathaniel Heintz Eric F. Schmidt |
| author_sort | Alexander R. Nectow |
| collection | DOAJ |
| description | Summary: Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. : Nectow et al. describe vTRAP, a technology to purify translating mRNAs from genetically defined cell types in a spatiotemporally restricted fashion. Multiplexing vTRAP with other technologies offers a comprehensive strategy to interrogate the precise role of individual, cell-type-specific genes in neural circuit function. Keywords: AAV, molecular profiling, TRAP, MCH, corticothalamic, Ntsr1, RNA-seq |
| first_indexed | 2024-12-14T22:44:00Z |
| format | Article |
| id | doaj.art-74f62f4da03d43449f074752f896fc74 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-14T22:44:00Z |
| publishDate | 2017-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-74f62f4da03d43449f074752f896fc742022-12-21T22:44:53ZengElsevierCell Reports2211-12472017-04-01193655667Rapid Molecular Profiling of Defined Cell Types Using Viral TRAPAlexander R. Nectow0Maria V. Moya1Mats I. Ekstrand2Awni Mousa3Kelly L. McGuire4Caroline E. Sferrazza5Bianca C. Field6Gabrielle S. Rabinowitz7Kirsty Sawicka8Yupu Liang9Jeffrey M. Friedman10Nathaniel Heintz11Eric F. Schmidt12Laboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Princeton Neuroscience Institute, Princeton University, Lot 20 Washington Road, Princeton, NJ 08544, USA; Corresponding authorLaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USALaboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USAHospital Informatics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USALaboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USALaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Corresponding authorLaboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Corresponding authorSummary: Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. : Nectow et al. describe vTRAP, a technology to purify translating mRNAs from genetically defined cell types in a spatiotemporally restricted fashion. Multiplexing vTRAP with other technologies offers a comprehensive strategy to interrogate the precise role of individual, cell-type-specific genes in neural circuit function. Keywords: AAV, molecular profiling, TRAP, MCH, corticothalamic, Ntsr1, RNA-seqhttp://www.sciencedirect.com/science/article/pii/S2211124717303947 |
| spellingShingle | Alexander R. Nectow Maria V. Moya Mats I. Ekstrand Awni Mousa Kelly L. McGuire Caroline E. Sferrazza Bianca C. Field Gabrielle S. Rabinowitz Kirsty Sawicka Yupu Liang Jeffrey M. Friedman Nathaniel Heintz Eric F. Schmidt Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP Cell Reports |
| title | Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP |
| title_full | Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP |
| title_fullStr | Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP |
| title_full_unstemmed | Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP |
| title_short | Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP |
| title_sort | rapid molecular profiling of defined cell types using viral trap |
| url | http://www.sciencedirect.com/science/article/pii/S2211124717303947 |
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