RNAi Core

RNAi核心

• 批准号: 7291580
• 负责人: WOJCIECH MICHAEL ZAWADA
• 金额: $ 34.82万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/7291580
• 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

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已经确定了大量候选基因，这些基因在选择性饲养的高或低酒精偏好小鼠的大脑中差异表达。在果蝇的酒精耐受性筛选中发现了额外的保守基因。操纵这些基因的表达自然成为研究的关键组成部分，旨在进一步确定它们在酒精偏好或耐受性发展中的作用。RNA干扰核心（RNAi Core）将为在选定和精确定义的大脑区域中系统修饰靶基因的表达提供一种手段。将采用两种技术平台进行体内RNA干扰：(1)小干扰RNA （siRNA）通过直接注射或输注进入中枢神经系统；(2)短发夹RNA （shRNA）通过慢病毒载体转导传递。首先，这些方法将使用在HAP和LAP小鼠中鉴定的促成酒精偏好饮酒行为的转录本进行标准化，并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研究者提供重要的资源，包括那些使用大鼠模型的研究者。在提交这份报告时，来自加州、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狂欢和依赖领域展开合作。该中心还将为INIA研究人员提供RNAi治疗，这些研究人员将随着更多靶基因的出现而开发对RNAi服务的需求。拟议的项目将由一个项目评价委员会进行评价，以便与该协会西部目标相结合，该委员会由该协会指导委员会成员和一名独立顾问组成。该核心将利用我们与Dharmacon公司的持续合作，后者是siRNA开发领域的领导者。达摩康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。核心目标将通过成功完成以下目标来实现：目标1。进行高通量的体外筛选RNAi序列针对感兴趣的基因。目标2。利用RNAi在精确的神经解剖靶点内沉默选定基因的体内表达。目标3。检查基因沉默对行为和转录的影响。RNAi核心的创建是INIA已经完成的工作的合理延伸。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操作，促进这些基因在酒精偏好中的功能研究。