In vivo Ribosome Tagging in Neuroendocrine Cell Types

神经内分泌细胞类型的体内核糖体标记

• 批准号: 7474503
• 负责人: PAUL S AMIEUX
• 金额: $ 22.93万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7474503
• 关键词: Acute; Alleles; Animals; Antibodies; Biological Models; Breeding; Cell Separation; Cells; Complex; Complex Mixtures; Cultured Cells; Databases; Dissociation; Engineering; Epitopes; Gene Expression Profile; Gene Expression Profiling; Genes; Genomics; Goals; Hela Cells; Hypothalamic structure; Individual; Lasers; Length; Methodology; Methods; Microarray Analysis; Microdissection; Molecular Profiling; Mouse Strains; Mus; Neuroendocrine Cell; Neurosecretory Systems; Partner in relationship; Perfusion; Physiological; Pituitary Gland; Polyribosomes; Population; Proteins; Reproduction; Ribosomal Proteins; Ribosomes; Running; Technology; Testing; Tissue Embedding; Tissues; Transcript; Transgenic Mice; Translating; base; cell type; embryonic stem cell; homologous recombination; hypothalamic pituitary gonadal axis; in vivo; insight; interest; internal control; male; novel; novel strategies; particle; promoter; recombinase; sample fixation; tissue processing; tool

DESCRIPTION (provided by applicant): The arrival over the past ten years of genomic tools for analyzing the transcriptome of key neuroendocrine tissues has provided considerable new insights into the complexities of the hypothalamic-pituitary-gonadal axis. These genomic approaches invariably depend upon acute isolation of cell types of interest from complex mixtures of many cell types using technologies like FACS analysis or Laser Cell Capture Microdissection. These approaches suffer from the need to significantly process the tissue containing the cell type of interest, including enzymatic dissociation of cells, perfusion, fixation, embedding of tissues for sectioning, and a variety of other manipulations that may alter the transcriptome of the cell type of interest. The ability to isolate the actively translated transcriptome from a cell type of interest in the context of a complex tissue, without the need for significant manipulation of the tissue of origin, would represent a significant advance in current genomic technology. In this proposal, we describe a novel strategy to rapidly and efficiently isolate the actively translated transcriptome by combining polysome analysis with mouse Cre- Lox technology and homologous recombination in embryonic stem cells. In this proposal, our goal is to develop lines of mice bearing a silent epitope-tagged allele of candidate ribosomal proteins. When bred to transgenic mice expressing Cre recombinase in specific neuroendocrine cell types, these silent alleles will express the epitope-tagged ribosomal proteins, which will be incorporated into actively translating ribosome particles, including polysomes. Polysomes will be immunoprecipitated using antibodies to the epitope tags and run on gradients to separate mRNAs with differing levels of translational efficiency. Low, medium, and highly translated mRNAs expressed in specific neuroendocrine cell types under varying physiological states can then be analyzed on microarrays followed by Quantitative-PCR.

描述（由申请人提供）：过去十年来的基因组工具的到来，用于分析关键神经内分泌组织的转录组，为下丘脑 - 垂体 - 长轴的复杂性提供了相当大的新见解。 这些基因组方法总是依赖于使用FACS分析或激光细胞捕获微分辨率等技术从许多细胞类型的复杂混合物中急性分离感兴趣的急性分离。 这些方法需要显着处理包含感兴趣的细胞类型的组织，包括细胞的酶解离，灌注，固定，组织嵌入进行切片的嵌入以及可能改变感兴趣细胞类型的转录组的其他各种操作。 在复杂组织的背景下，不需要对原始组织进行重大操纵的情况下，将主动翻译的转录组与兴趣的细胞类型分离的能力将代表当前基因组技术的重大进展。 在此提案中，我们描述了一种新的策略，可以通过将多元体分析与小鼠旋转技术和胚胎干细胞中的同源重组相结合，以快速有效地分离主动翻译的转录组。 在此提案中，我们的目标是开发带有候选核糖体蛋白质静音表位等位基因的小鼠线。 当繁殖到在特定神经内分泌细胞类型中表达CRE重组酶的转基因小鼠时，这些无声等位基因将表达表位标记的核糖体蛋白，该核糖体蛋白将掺入积极地翻译核糖体颗粒，包括多聚体。 多聚体将使用对表位标签的抗体进行免疫沉淀，并以梯度运行以分离具有不同平移效率水平的mRNA。 然后可以在微阵列上分析在不同生理状态下在特定神经内分泌细胞类型中表达的低，中和高度翻译的mRNA，然后在微阵列上进行定量PCR。