Cell Type Specific Analysis of MiRNA Expression

miRNA 表达的细胞类型特异性分析

• 批准号: 8806621
• 负责人: Edgardo Rodriguez
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8806621
• 关键词: Adult; Affect; Behavioral Symptoms; Benchmarking; Binding Proteins; Biochemical; Brain; Brain Diseases; CAG repeat; Cataloging; Catalogs; Cells; Cerebellum; Communities; Complex; Corpus striatum structure; Data Analyses; Development; Disease; Dopamine Receptor; Dose; Engineering; Excision; Family; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Expression Regulation; Gene Targeting; Gene Transfer; Genes; Genetic; Goals; Health; High-Throughput Nucleotide Sequencing; High-Throughput RNA Sequencing; Knock-in Mouse; Lead; Libraries; Maintenance; Mediating; Methods; MicroRNAs; Modeling; Molecular; Mus; Neurons; Pathogenesis; Pathogenicity; Physiological; Play; Population; Publishing; Purkinje Cells; RNA Sequence Analysis; RNA library; Recombinant adeno-associated virus (rAAV); Reproducibility; Research; Role; Route; Small RNA; Specificity; Technology; Testing; Tissues; Toxic effect; Transgenic Mice; Type 1 Spinocerebellar Ataxia; Untranslated RNA; Virus; base; cell type; cost; deep sequencing; member; miRNA expression profiling; mouse model; nervous system disorder; neurotoxicity; novel; novel strategies; novel therapeutics; polyglutamine; programs; recombinase; research study; selective expression; tool

DESCRIPTION (provided by applicant): A number of neurological disorders are characterized by the preferential dysfunction and/or loss of specific neuronal cell populations and the mechanisms underlying this cell type specific vulnerability remain poorly understood. It has been proposed that the differential neuronal vulnerability observed in these disorders arises from disruptions in cell type specific gene expression regulatory programs responsible for maintaining the physiological identity and function of the affected neurons. MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression networks at the posttranscriptional level. Studies, including ours, have implicated altered miRNA expression in the pathogenicity of several neurological disorders. Here, we propose the development of a novel molecular approach to investigate the role that neural cell type specific changes in miRNA function play in the pathogenicity of neurological diseases. The approach relies on a recombinant adeno-associated virus (rAAV)-based, Cre recombinase-dependent genetic Flp-excision switch (FLEX switch) that restricts the expression of an ectopically delivered miRNA-binding protein Argonaute-2 to Cre expressing cells. We call this approach miFLAGO (miRNA-associated, FLEX switch regulated, AGO2). Experiments in Aim-1 will validate the functionality and reproducibility of miFLEX in the cerebellum of adult transgenic mice engineered to selectively express Cre recombinase in Purkinje cells. Purkinje cell-specific miFAGO-miRNA complexes will be isolated and used to build miRNA libraries for high-throughput RNA sequencing analysis. In Aim-2, we will use miFLAGO to investigate the role that cell type specific changes in miRNA function play in the pathogenicity of a mouse model of Spinocerebellar ataxia type-1, a polyglutamine disorder caused by the expansion of a CAG repeat in the ATXN1 gene. The short-term goal of this proposal is to provide the research community with a validated novel molecular tool for the study of cell type specific miRNA function. Long-term, we aim to fine-tune our understanding of neural cell type specific gene expression regulatory programs and how they might mediate differential vulnerability in neurological disease, with the goal of identifying novel therapeutic routes.

描述（由申请人提供）：许多神经系统疾病的特征在于特定神经元细胞群的优先功能障碍和/或损失，并且对这种细胞类型特异性脆弱性的机制仍然知之甚少。有人提出，在这些疾病中观察到的差异神经元脆弱性来自负责维持受影响神经元的生理特性和功能的细胞类型特异性基因表达调控程序的破坏。microRNA（miRNAs）是一类在转录后水平参与调控基因表达网络的非编码小RNA。包括我们在内的研究表明，miRNA表达的改变与几种神经系统疾病的致病性有关。在这里，我们提出了一种新的分子方法的发展，调查的作用，神经细胞类型的特异性变化的miRNA功能在神经系统疾病的致病性。该方法依赖于基于重组腺相关病毒（rAAV）的Cre重组酶依赖性遗传Flp切除开关（FLEX开关），其将异位递送的miRNA结合蛋白Argonaute-2的表达限制于Cre表达细胞。我们称这种方法为miFLAGO（miRNA相关，FLEX开关调节，AGO 2）。Aim-1中的实验将验证miFLEX在成年转基因小鼠小脑中的功能性和再现性，所述成年转基因小鼠经工程改造以在浦肯野细胞中选择性表达Cre重组酶。浦肯野细胞特异性miFAGO-miRNA复合物将被分离并用于构建miRNA文库以进行高通量RNA测序分析。在目的-2中，我们将使用miFLAGO来研究细胞类型特异性的miRNA功能变化在脊髓小脑共济失调1型小鼠模型的致病性中所起的作用，脊髓小脑共济失调1型是一种由ATXN 1基因中CAG重复序列扩增引起的多聚谷氨酰胺疾病。该提案的短期目标是为研究界提供一种经过验证的新型分子工具，用于研究细胞类型特异性miRNA功能。长期而言，我们的目标是微调我们对神经细胞类型特异性基因表达调控程序的理解，以及它们如何介导神经系统疾病的差异脆弱性，目的是确定新的治疗途径。