Site-specific Gene Silencing by RNA Interference

描述（由申请人提供）：本申请的目的是开发RNA干扰（RNAi）作为一种完全沉默中枢神经系统基因表达的新工具。具体而言，我们将确定使用RNAi作为一种以空间和时间控制的方式消除基因表达的选择方法的可行性，并确定对基因功能和动物行为的下游影响。与传统方法（敲除、病变、拮抗剂和反义）相比，这种方法具有明显的优势，因为它提供了更高的特异性和效力。自从偶然发现双链RNA （dsRNA）以一种表型上类似于基因敲除的方式作为一种极其特异性和有效的基因表达抑制剂以来，利用RNAi来阐明模型系统（如秀丽隐杆线虫、果蝇和培养细胞）中基因功能的研究激增。然而，其在哺乳动物体内的应用非常有限。因此，本研究的目的是：(1)建立并优化RNAi在大鼠脑中的作用剂量、作用时间等关键参数。为此，我们选择促肾上腺皮质激素释放因子（CRF）作为模型神经肽，下丘脑室旁核作为模型位点。我们将通过立体定向手术将dsRNA引入PVN。特异性沉默PVN中CRF对神经解剖学和基因表达的影响将通过原位和免疫组织化学研究来确定。对下丘脑-垂体-肾上腺（HPA）轴功能的下游影响将通过使用放射免疫测定法确定直接受CRF影响的基础和应激激活参数的变化来评估，如促肾上腺皮质激素（ACTH）和皮质酮(B)。(2)我们将评估这些优化参数是否可以更广泛地应用于抑制另一个表达CRF的大脑部位（杏仁核）的CRF表达。杏仁核是大脑中涉及恐惧/焦虑反应的主要部位。我们将通过测量基础和应激激活的ACTH和b的变化来确定杏仁核CRF在急性约束应激反应中的作用。此外，我们将确定几种基因和神经肽的RNA和蛋白质表达水平的变化，如CRF、加压素、丙氨酸、尿皮质素III和c-fos。虽然RNAi在基础研究中的应用非常重要，但一旦建立，它在解剖神经回路方面具有巨大的应用潜力，并可能应用于沉默神经退行性疾病相关基因的表达

DESCRIPTION (provided by applicant): The objective of this application is to develop RNA interference (RNAi) as a novel tool to completely silence gene expression in the central nervous system. Specifically, we will determine the feasibility of using RNAi as a method of choice to eliminate gene expression in a spatially- and temporally-controlled manner, and determine downstream effects on gene function and animal behavior. This approach will offer distinct advantage over conventional methods (knockouts, lesions, antagonists and antisense) by providing increased specificity and potency. Ever since the serendipitous discovery that double stranded RNA (dsRNA) functions as an extremely specific and potent inhibitor of gene expression in a manner phenotypically akin to a genetic knockout, there has been a surge in studies using RNAi to elucidate gene function in model systems such as the C. elegans, drosophila, and cultured cells. However, its application in vivo in mammals has been very limited. Thus, the aims of this study are to (1) establish and optimize the key parameters such as the dose and time course of RNAi action in rat brain. Towards this end, we have selected corticotropin-releasing factor (CRF) as the model neuropeptide and the hypothalamic paraventricular nucleus as the model locus. We will introduce dsRNA into the PVN using stereotaxic surgery. Effect of specifically silencing CRF in the PVN on neuroanatomy and gene expression will be determined by in situ and immunohistochemical studies. Downstream effect on hypothalamic-pituitary adrenal (HPA) axis function will be assessed by determining changes in basal and stress activated parameter directly influenced by CRF such as adrenocorticotropin hormone (ACTH) and corticosterone (B) using radioimmunoassays. (2) We will assess whether these optimized parameter can be applied more globally to inhibit CRF expression in another brain site (amygdala) that expresses CRF. Amygdala is the principal brain locus involved in fear/anxiety responses. We will determine role of amygdala CRF on acute restraint stress responses by measuring changes in basal and stress activated changes in ACTH and B. Furthermore, we will determine changes in expression levels of both RNA and protein of several genes and neuropeptides such as the CRF, vasopressin, galanin, urocortin III and c-fos. While application of RNAi in basic research is of vast importance, once established, it holds tremendous potential for its applicability in dissecting out neural circuitry and may be applied for silencing expression of genes involved in neurodegenerative diseases