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

Q2 Neuroscience Current Protocols in Neuroscience Pub Date : 2020-03-05 DOI:10.1002/cpns.93
Vivek Mahadevan, Areg Peltekian, Chris J. McBain
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引用次数: 3

摘要

gaba能中间神经元包括哺乳动物大脑中一个小而多样的神经元子集,它们严格调节神经元回路的成熟和信息流,并最终调节行为。由于它们在许多神经系统疾病的病因学中处于中心地位,因此在不同生理情况下检查这些神经元的分子结构引起了更广泛的神经科学界的兴趣。在过去的几年里,下一代测序(NGS)方法出现了爆炸式增长,旨在识别神经元多样性中的遗传和状态依赖亚型。尽管采用了几种方法来解决神经元分子多样性,核糖体标记已经出现在识别神经元亚型翻译体的前沿。这种方法主要依赖于Cre重组酶驱动的血凝素A (HA)标记的RiboTag小鼠在感兴趣的神经元亚型中的表达。这允许细胞类型特异性,核糖体参与mRNA的免疫沉淀,在体细胞和神经元过程中表达,用于靶向定量实时PCR (qRT-PCR)或高通量RNA测序分析。在这里,我们详细介绍了成功应用RiboTag技术分析中枢神经系统中gaba能中间神经元以及理论上其他稀疏细胞类型的典型技术注意事项。2020年出版。美国政府。基本方案1:培养小鼠获得RiboTag纯合子支持方案1:检测异位Cre重组酶表达基本方案2:检测RiboTag检测支持方案2:实时定量PCR (qRT-PCR)检测RiboTag衍生的细胞类型特异性rna支持方案3:构建细胞类型特异性RNA-seq文库支持方案4:对RiboTag衍生的RNA-seq数据集进行二次分析
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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

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Current Protocols in Neuroscience
Current Protocols in Neuroscience Neuroscience-Neuroscience (all)
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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.
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