RNAi Core

描述（由申请人提供）：最初的五年INIA West已经确定了大量的候选基因，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鉴定了其他保守基因。操纵这些基因的表达自然成为旨在进一步确定其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）将提供一种在选定和精确定义的大脑区域中系统修饰靶基因表达的方法。将采用两种用于体内RNA干扰的技术平台：（1）通过直接注射或输注到CNS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，这些方法将使用在HAP和HAP小鼠中鉴定为有助于酒精偏好饮酒行为的转录物进行标准化，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研究人员提供重要的资源，包括那些使用大鼠模型的研究人员。在提交本报告时，来自加州、科罗拉多、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开展合作。核心还将为INIA研究人员进行RNAi治疗，他们将随着其他靶基因的出现而发展对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西部目标相结合。核心将利用我们与Dharmacon公司的持续合作，Dharmacon公司是siRNA开发领域的领导者。Dharmacon将提供许多必要的试剂，并与我们合作提高CNS中基因沉默的效率。核心的目标将通过成功完成以下目标来实现：目标1。对靶向目标基因的RNAi序列进行高通量体外筛选。 目标2.在精确的神经解剖学靶点内采用RNAi沉默体内选定基因的表达。目标3：研究基因沉默的行为和转录效应。RNAi核心的创建是INIA已经完成的工作的逻辑延伸。该核心将允许系统和高通量操纵哺乳动物中枢神经系统中的基因，促进这些基因在酒精偏好的功能研究。

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.