RiboTag: A novel technique to profile cell type specific gene expression and inv

RiboTag：一种分析细胞类型特异性基因表达和反转录的新技术

• 批准号: 8473919
• 负责人: George STANLEY MCKNIGHT
• 金额: $ 35.95万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8473919
• 关键词: Address; Aging; Antibodies; Biochemical; Biochemical Genetics; Biological Assay; Brain; Cell Nucleus; DNA Sequence; Disease; Environment; Epitopes; Event; Gene Expression; Genes; Genetic Techniques; Genetic Translation; Goals; Hormones; Immunologic Techniques; Immunoprecipitation; In Situ Hybridization; Individual; Lasers; Learning; Measures; Memory; Messenger RNA; Methods; Microarray Analysis; Molecular Profiling; Mouse Strains; Mus; Nervous system structure; Neuraxis; Neuromodulator; Neurons; Neurosciences; Neurotransmitters; Occupations; Organism; Pharmaceutical Preparations; Physiological; Polyribosomes; Population; Protein Isoforms; Proteins; RNA; Regulation; Reporter; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Specificity; Speed; Substance Addiction; Substance abuse problem; Synapses; Synaptic plasticity; Techniques; Technology; Testing; Time; Transgenic Organisms; Translating; Translational Regulation; cell type; drug of abuse; improved; in vivo; nervous system disorder; new technology; next generation; novel; post-traumatic stress; protein expression; recombinase; response; tool; transcriptome sequencing

Project Summary The brain is composed of hundreds of different neuronal subtypes that each have unique jobs to do. One of the most significant long-term goals of neuroscience is to understand how these neuronal subtypes do their job by producing specific proteins, contacting other neurons, and changing in response to physiological and pathological contexts. Unfortunately, techniques to capture the total translated mRNA from a defined subtype of neurons as the organism responds to the environment, hormones, drugs, and other regulators are lacking. This proposal addresses that need with a novel biochemical and genetic technique to express epitope-tagged ribosomal proteins in response to a cell type specific Cre recombinase in mouse brain. The polyribosomes containing the transcribed and translatable mRNAs are isolated by immunological techniques and then the RNA is analyzed by PCR, microarray, and next-generation DNA sequencing. Our specific aims are to: (1) Develop techniques to optimize polyribosome immunoprecipitation from brain using the RiboTag mouse that has already been created with an HA-tagged Rpl22 ribosomal protein under Cre recombinase regulation. (2) Create a new RiboTag mouse line with a Flag-tagged Rpl23a that will respond to Cre recombinase activation. (3) Activate Rpl22-HA and Rpl23a- Flag with specific Cre recombinase transgenics to test the ability of the RiboTag isolation technique to detect neuron specific mRNAs and the changes that occur under in vivo physiological regulation. (4) Examine whether the RiboTag techniques differentiate between translated mRNAs and those that are translationally repressed. These novel mouse strains and biochemical methods should allow neuroscientists to measure changes in gene expression and translational regulation with greater resolution and higher throughput than previously available and speed our understanding of the underlying changes in RNA and protein that determine synaptic plasticity. Relevance In order to understand how the brain adapts to a changing environment under physiological circumstances or how specific neuronal populations degenerate or malfunction in disease, we must have robust and widely available techniques to measure gene and protein expression in specific neuronal subtypes. The ribosome tagging (RiboTag) technology we are developing will provide such a tool to address questions about gene expression and mRNA translation during adaptive changes in the brain-changes that occur during memory and learning, substance abuse and addiction, aging, and in response to neurological disorders.

项目摘要 大脑由数百种不同的神经元亚型组成，每种亚型都有 独一无二的工作要做。神经科学最重要的长期目标之一是 为了了解这些神经元亚型是如何通过产生特定的 蛋白质，接触其他神经元，并对生理变化作出反应 以及病态的背景。不幸的是，捕捉总数的技术 当生物体对特定的神经元亚型做出反应时，翻译出的mRNA 环境、激素、药物和其他监管机构都缺乏。这 提案通过一种新的生化和基因技术满足了这一需求 表达表位标记的核糖体蛋白对细胞类型特异性Cre的反应 重组酶在小鼠脑中的表达。含有转录和转录的多聚核糖体 通过免疫学技术分离出可翻译的mRNAs，然后将rna 通过聚合酶链式反应、微阵列和下一代DNA测序进行分析。我们的 具体目标是：(1)开发优化多核糖体的技术 用已有的RiboTag小鼠进行脑免疫沉淀 用透明质酸标记的Rpl22核糖体蛋白在Cre重组酶下产生 监管。(2)创建一个新的RiboTag小鼠品系，该品系带有Flag标记的Rpl23a 会对Cre重组酶的激活做出反应。(3)激活Rpl22-HA和Rpl23a- 带有特定Cre重组酶转基因的FLAG以测试RiboTag的能力 检测神经元特异性mRNAs的分离技术及其发生的变化 在体内的生理调节下。(4)检查RiboTag是否 翻译的mRNA和被翻译的mRNA之间的技术区别 翻译上被压抑。这些新的小鼠品系和生化方法 应该允许神经科学家测量基因表达的变化和 转换调节具有更高的分辨率和更高的吞吐量 并使我们更快地了解 决定突触可塑性的RNA和蛋白质。 相关性 为了了解大脑是如何适应不断变化的环境的 生理环境或特定神经元群体如何退化或 疾病中的故障，我们必须有强大和广泛可用的技术来 测量特定神经元亚型的基因和蛋白表达。这个 我们正在开发的核糖体标记(RiboTag)技术将提供这样一种 解决有关基因表达和信使核糖核酸翻译问题的工具 大脑的适应性变化--在记忆和学习过程中发生的变化， 药物滥用和成瘾，衰老，以及对神经紊乱的反应。