RNAi Core

描述(申请人提供)：最初的五年INIA West发现了大量候选基因，这些基因在选择性培育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查中发现了其他保守基因。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)将为在选定和精确定义的大脑区域系统地修改目标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)通过直接注射或输注到中枢神经系统传递的小干扰RNA(SiRNA)；(2)通过慢病毒载体转导传递的短发夹状RNA(ShRNA)。最初，这些方法将使用在HAP和LAP小鼠中确定的有助于酒精偏好饮酒行为的转录来标准化，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调查人员，包括那些从事大鼠模型工作的人员提供重要的资源。在提交本报告时，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狂欢和依赖领域进行合作。该核心还将为INIA调查人员进行RNAi治疗，随着更多目标基因的出现，他们将发展对RNAi服务的需求。拟议的项目将由一个项目评价委员会对其与INIA西部目标的结合进行评价，该委员会由INIA指导委员会成员和一名独立顾问组成。核心将利用我们与达哈拉康公司的持续合作，达哈马克公司是siRNA开发领域的领先企业。达哈纳康将提供许多必要的试剂，并与我们合作，提高中枢神经系统中基因沉默的效率。该核心的目标将通过成功完成以下目标来实现：目标1.进行针对目的基因的RNAi序列的体外高通量筛选。目的2.在精确的神经解剖学靶点内利用RNAi技术在体内沉默所选基因的表达。目的3.检测基因沉默的行为和转录效应。RNAi核心的建立是INIA已经完成的工作的合乎逻辑的延伸。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。

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.