RNAi Core

RNAi核心

基本信息

批准号：
7483232
负责人：
Paula Hoffman
金额：
$ 34.12万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7483232
关键词：
Acute Adrenergic Agents Affect Alcohol consumption Alcohols Amygdaloid structure Animal Model Animals Antibodies Area Behavioral Binding Biochemical Genetics Boutos Brain Brain region Breeding C57BL/6 Mouse California Candidate Disease Gene Cell Line Cell Nucleus Cell Transplants Cerebral Ventricles Chronic Classification Collaborations Colorado Complex Condition Consumption D Aspartate Databases Dependence Development Dissection Drosophila genus Ensure Ethanol Evaluation Exhibits Figs - dietary Funding Future Gene Expression Gene Expression Profile Gene Mutation Gene Silencing Gene Targeting Genes Genetic Genetic Determinism Genetic Predisposition to Disease Genetic Screening Genetic Transcription Genome Glutamate Receptor Glutamates Goals Half-Life Heavy Drinking Homer protein Homologous Gene Immunoblotting In Situ Hybridization In Vitro Indiana Individual Industry Infusion procedures Injection of therapeutic agent Knock-out Knowledge Laboratories Lentivirus Vector Letters Lipids Localized Magic Manuals Maps Metabotropic Glutamate Receptors Methodology Methods Microinjections Modality Modeling Modification Molecular Mouse Strains Mus N-Methyl-D-Aspartate Receptors Nature Neuroblastoma Numbers Oregon Other Genetics Outcome Pathway interactions Peer Review Phenotype Play Polymerase Chain Reaction Principal Investigator Procedures Production Protein Overexpression Proteins Pump Quantitative Trait Loci RNA RNA Interference Rattus Reagent Regulation Research Research Design Research Personnel Resources Reverse Transcriptase Polymerase Chain Reaction Rodent Model Role Scaffolding Protein Schedule Screening procedure Serotonin Services Shipping Ships Signal Transduction Site Site-Directed Mutagenesis Small Interfering RNA Solutions Standards of Weights and Measures Synapses Synaptic Transmission Tars Testing Texas Time Transcript Transfection Translations Up-Regulation Validation Viral Viral Vector Week Western Blotting Withdrawal Work adrenergic alcohol exposure alcohol sensitivity base behavior test binge drinking brain tissue cell type cytokine day drinking drinking behavior experience high throughput analysis immunocytochemistry improved in vivo infancy interest intracellular protein transport knock-down laser capture microdissection man member mouse model nerve stem cell postsynaptic preference programs protein transport research study small hairpin RNA tissue processing tool trafficking transmission process urocortin vector

项目摘要

DESCRIPTION (provided by applicant): The original five-year INIA West has identified a large number of candidate genes that are differentially expressed in the brains of mice selectively bred for either high- or low-preference for alcohol. Additional conserved genes were identified in screens for alcohol tolerance in Drosophila. Manipulating the expression of these genes has naturally become a critical component of studies designed to further define their role in the development of alcohol preference or tolerance. The RNA Interference Core (RNAi Core) will provide a means for systematic modification of the target genes' expression in selected and precisely-defined brain areas. Two technological platforms for in vivo RNA interference will be employed: (1) small interfering RNA (siRNA) delivered by a direct injection or infusion into the CNS and (2) short hairpin RNA (shRNA) delivered via a lentiviral vector-based transduction. Initially, these methods will be standardized using transcripts identified in HAP and LAP mice as contributing to alcohol preference drinking behavior, and the functional consequences of the RNAi treatment will be assessed in these mice. The successful implementation of the methodology will provide an important resource for all INIA investigators, including those working with rat models. At the time of this submission, laboratories from California, Colorado, Indiana, Oregon and Texas are already collaborating in both Binge and Dependence Domains. The core will also perform RNAi treatments for INIA investigators, who will develop need for RNAi services with the emergence of additional target genes. Proposed projects will be evaluated for integration with INIA West goals by a Project Evaluation Committee composed of members of the INIA Steering Committee and an independent consultant. The core will capitalize on our ongoing collaboration with Dharmacon Corporation, a leader in the field of siRNA development. Dharmacon will provide many of the necessary reagents and work with us on improving the efficiency of gene silencing in the CNS. The core's goals will be accomplished by successful completion of the following aims: Aim 1. Perform high throughput in vitro screening of the RNAi sequences targeting the genes of interest. Aim 2. Silence expression of selected genes in vivo employing RNAi within precise neuroanatomical targets. Aim 3. Examine behavioral and transcriptional effects of gene silencing. The creation of the RNAi core is a logical extension of the work already completed by the INIA. The core will allow for systematic and high throughput manipulation of genes in the mammalian CNS, facilitating functional studies of these genes in alcohol preference.

描述（由申请人提供）：最初为期五年的 INIA West 已鉴定出大量候选基因，这些基因在选择性饲养的高酒精偏好或低酒精偏好小鼠的大脑中差异表达。在果蝇酒精耐受性筛选中鉴定出其他保守基因。操纵这些基因的表达自然成为旨在进一步确定它们在酒精偏好或耐受性发展中的作用的研究的关键组成部分。 RNA 干扰核心 (RNAi Core) 将提供一种方法，系统地修改选定且精确定义的大脑区域中目标基因的表达。将采用两种体内RNA干扰技术平台：（1）通过直接注射或输注到中枢神经系统中递送的小干扰RNA（siRNA）和（2）通过基于慢病毒载体的转导递送的短发夹RNA（shRNA）。最初，这些方法将使用 HAP 和 LAP 小鼠中鉴定的转录本进行标准化，因为这些转录本有助于酒精偏好的饮酒行为，并且将在这些小鼠中评估 RNAi 治疗的功能后果。该方法的成功实施将为所有 INIA 研究人员（包括那些研究大鼠模型的人员）提供重要资源。在提交本文时，来自加利福尼亚州、科罗拉多州、印第安纳州、俄勒冈州和德克萨斯州的实验室已经在 Binge 和依赖领域进行合作。该核心还将为 INIA 研究人员进行 RNAi 治疗，随着其他靶基因的出现，他们将开发对 RNAi 服务的需求。由 INIA 指导委员会成员和一名独立顾问组成的项目评估委员会将评估拟议项目是否与 INIA West 目标相结合。该核心将利用我们与 siRNA 开发领域的领导者 Dharmacon Corporation 的持续合作。 Dharmacon 将提供许多必要的试剂，并与我们合作提高中枢神经系统基因沉默的效率。该核心目标将通过成功完成以下目标来实现：目标 1. 对针对感兴趣基因的 RNAi 序列进行高通量体外筛选。目标 2. 在精确的神经解剖靶标内采用 RNAi 沉默体内选定基因的表达。目标 3. 检查基因沉默的行为和转录效应。 RNAi 核心的创建是 INIA 已完成工作的逻辑延伸。该核心将允许对哺乳动物中枢神经系统中的基因进行系统和高通量的操作，促进这些基因在酒精偏好中的功能研究。