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

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 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，并响应生理变化， 和病理学背景。不幸的是， 当生物体响应时， 缺乏环境，激素，药物等调节剂。这 该提案提出了一种新的生物化学和遗传技术， 表达表位标记的核糖体蛋白，以响应细胞类型特异性Cre 重组酶。多聚核糖体含有转录的和 通过免疫学技术分离可翻译的mRNA， 通过PCR、微阵列和下一代DNA测序进行分析。我们 具体目标是：（1）开发优化多聚核糖体的技术 使用RiboTag小鼠从大脑中进行免疫沉淀， 在Cre重组酶下用HA标记的Rp 122核糖体蛋白产生 调控(2)创建一个新的RiboTag鼠标线与标志标记的Rpl 23 a， 会对Cre重组酶的激活做出反应(3)激活Rpl 22-HA和Rpl 23 a- 用特异性Cre重组酶转基因标记以测试RiboTag的能力 分离技术来检测神经元特异性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 压抑的情绪。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， 翻译调控具有更高的分辨率和更高的吞吐量， 以前可用的，并加快我们对潜在变化的理解， 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环境变化的， 生理环境或特定的神经元群体如何退化， 我们必须有强大的和广泛可用的技术， 测量特定神经元亚型中的基因和蛋白质表达。的 我们正在开发的核糖体标记（RiboTag）技术将提供这样一种 一个解决基因表达和mRNA翻译问题的工具， 大脑中的适应性变化--发生在记忆和学习过程中的变化， 药物滥用和成瘾、衰老以及对神经系统疾病的反应。

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.