{"title":"条件核糖体标记在gaba能中间神经元和其他稀疏细胞类型中的翻译组分析","authors":"Vivek Mahadevan, Areg Peltekian, Chris J. McBain","doi":"10.1002/cpns.93","DOIUrl":null,"url":null,"abstract":"<p>GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase–driven expression of hemagglutinin A (HA)–tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government.</p><p><b>Basic Protocol 1</b>: Breeding mice to obtain RiboTag homozygosity</p><p><b>Support Protocol 1</b>: Detection of ectopic Cre recombinase expression</p><p><b>Basic Protocol 2</b>: The RiboTag assay</p><p><b>Support Protocol 2</b>: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA</p><p><b>Support Protocol 3</b>: Construction of cell-type-specific RNA-seq library</p><p><b>Support Protocol 4</b>: Secondary analyses of RiboTag-derived RNA-seq dataset</p>","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.93","citationCount":"3","resultStr":"{\"title\":\"Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types\",\"authors\":\"Vivek Mahadevan, Areg Peltekian, Chris J. 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This approach primarily relies on Cre recombinase–driven expression of hemagglutinin A (HA)–tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government.</p><p><b>Basic Protocol 1</b>: Breeding mice to obtain RiboTag homozygosity</p><p><b>Support Protocol 1</b>: Detection of ectopic Cre recombinase expression</p><p><b>Basic Protocol 2</b>: The RiboTag assay</p><p><b>Support Protocol 2</b>: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA</p><p><b>Support Protocol 3</b>: Construction of cell-type-specific RNA-seq library</p><p><b>Support Protocol 4</b>: Secondary analyses of RiboTag-derived RNA-seq dataset</p>\",\"PeriodicalId\":40016,\"journal\":{\"name\":\"Current Protocols in Neuroscience\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2020-03-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpns.93\",\"citationCount\":\"3\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current Protocols in Neuroscience\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpns.93\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"Neuroscience\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Neuroscience","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpns.93","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Neuroscience","Score":null,"Total":0}
Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types
GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase–driven expression of hemagglutinin A (HA)–tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government.
Basic Protocol 1: Breeding mice to obtain RiboTag homozygosity
Support Protocol 1: Detection of ectopic Cre recombinase expression
Basic Protocol 2: The RiboTag assay
Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA
Support Protocol 3: Construction of cell-type-specific RNA-seq library
Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset
期刊介绍:
Current Protocols in Neuroscience is a one-stop resource for finding and adapting the best models and methods for all types of neuroscience experiments. Updated every three months in all formats, CPNS is constantly evolving to keep pace with the very latest discoveries and developments. A year of these quarterly updates is included in the initial CPNS purchase price.