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）作为完全沉默中枢神经系统中基因表达的新工具。具体来说，我们将确定使用RNAi作为选择方法的可行性，以空间和时间控制的方式消除基因表达，并确定对基因功能和动物行为的下游影响。这种方法将通过提供更高的特异性和效力而比传统方法（敲除、损伤、拮抗剂和反义）具有明显的优势。自从偶然发现双链RNA（dsRNA）以表型上类似于基因敲除的方式作为基因表达的极其特异和有效的抑制剂起作用以来，使用RNAi来阐明模型系统（例如C.线虫、果蝇和培养细胞。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）建立和优化关键参数，如剂量和时间过程的RNAi作用在大鼠脑。为此，我们选择促肾上腺皮质激素释放因子（CRF）作为模型神经肽和下丘脑室旁核作为模型位点。我们将使用立体定位手术将dsRNA引入PVN。将通过原位和免疫组织化学研究确定PVN中特异性沉默CRF对神经解剖学和基因表达的影响。通过放射免疫测定法测定受CRF直接影响的基础和应激激活参数（如促肾上腺皮质激素（ACTH）和皮质酮（B））的变化，评估对下丘脑-垂体肾上腺（HPA）轴功能的下游影响。(2)我们将评估这些优化的参数是否可以更全面地应用于抑制CRF在另一个表达CRF的大脑部位（杏仁核）的表达。杏仁核是参与恐惧/焦虑反应的主要大脑部位。我们将通过测量ACTH和B的基础变化和应激激活变化来确定杏仁核CRF在急性束缚应激反应中的作用。此外，我们将确定几个基因和神经肽，如CRF，加压素，甘丙肽，urocortin III和c-fos的RNA和蛋白质表达水平的变化。虽然RNAi在基础研究中的应用非常重要，但一旦建立，它在解剖神经回路中的适用性方面具有巨大的潜力，并可用于沉默参与神经退行性疾病的基因的表达

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