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: 8292186
• 负责人: PAUL S AMIEUX
• 金额: $ 37.45万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292186
• 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; Stress; 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; protein expression; recombinase; response; tool

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 独特的工作要做。神经科学最重要的长期目标之一是 了解这些神经元亚型如何通过生产特定的 蛋白质，联系其他神经元，并响应生理 和病理环境。不幸的是，捕获总数的技术 随着生物的反应，从定义的神经元中定义的亚型翻译mRNA 缺乏环境，荷尔蒙，药物和其他监管机构。这 提案通过一种新颖的生化和遗传技术来解决需要 响应细胞类型特异性CRE的表达表位标记的核糖体蛋白 小鼠大脑中的重组酶。包含抄录的多核糖体和 可翻译的mRNA通过免疫学技术分离，然后是RNA 通过PCR，微阵列和下一代DNA测序分析。我们的 具体目的是：（1）开发技术以优化多核糖体 使用已经存在的Ribotag小鼠对大脑的免疫沉淀 用标有HA标记的RPL22核糖体蛋白在CRE重组酶下创建 规定。 （2）使用标记标记的RPL23A创建新的Ribotag鼠标线 将响应CRE重组酶激活。 （3）激活RPL22-HA和RPL23A-- 具有特定CRE重组酶转基因的标志以测试Ribotag的能力 隔离技术检测神经元特异性mRNA和发生的变化 在体内生理调节下。 （4）检查Ribotag是否 技术区分翻译的mRNA和那些的技术 翻译压抑。这些新型的小鼠菌株和生化方法 应该允许神经科学家衡量基因表达的变化和 转化调节，分辨率更高，吞吐量更高 以前可用并加快我们对基础变化的理解 确定突触可塑性的RNA和蛋白质。 关联 为了了解大脑如何适应不断变化的环境 生理环境或特定神经元种群如何退化或 疾病故障，我们必须具有强大且可广泛可用的技术 测量特定神经元亚型中的基因和蛋白质表达。这 我们正在开发的核糖体标记（Ribotag）技术将提供这样的 解决有关基因表达和mRNA翻译问题的工具 记忆和学习过程中发生的大脑变化的自适应变化， 药物滥用和成瘾，衰老以及对神经系统疾病的反应。