CCR RNAi initiative: High through put validation of siRN

通过RNA干扰(RNAi)在哺乳动物细胞中进行基因沉默已经成为研究生物过程的宝贵工具。RNAi是通过小的21个核苷酸的双链RNA，称为小干扰RNAs(SiRNAs)的作用，形成内源性酶复合体的一部分，称为RNA诱导沉默复合体(RISC)。更具体地说，siRNA双链的单链被选择性地装载到RISC中，在那里它为互补mRNA转录本的催化切割提供严格的指导。我们正在利用RNAi来探索与癌症相关的途径，包括验证抗癌靶点，潜在的识别新的靶点，开发新的模型系统，以及阐明/验证癌症相关表型的整体成分。为了获得大规模的合成siRNA资源，GSS与Qiagen Inc.达成了一项研究合作协议。到目前为止，Qiagen已经设计和合成了对应于大约400个基因的siRNA。为了充分利用这些资源，我们开发了一种96孔板格式的高通量自动化合成siRNA-脂类“反向转染法”(BioRobot 8000，Qiagen Inc.)。到目前为止，我们已经检测了对应于131个人类基因的267个siRNA介导的基因敲除。在这些siRNA中，超过70%的siRNAs在研究中的基因表达的稳态水平有统计上的显著下降。我们正在积极调查一些siRNA未能沉默的原因。在许多情况下，我们怀疑未能诱导沉默通常反映了与所讨论基因的不同可选转录本的表达有关的问题，以及阻止siRNA-RISC复合体切割的单核苷酸多态。在少数病例中，我们还研究了siRNAs的特异性。除了验证之外，我们现在正在扩展这个平台，使我们能够通过开发组合文库方法来进一步开展我们的药用基因组研究。

Gene silencing in mammalian cells through RNA interference (RNAi) has become an invaluable tool for the study of biological processes. RNAi is mediated through the action of small 21 nucleotide duplex RNAs, known as small interfering RNAs (siRNAs), which form part of an endogenous enzyme complex, termed the RNA-induced silencing complex (RISC). More specifically, a single strand of the siRNA duplex is selectively loaded into RISC where it provides stringent guidance for the catalytic cleavage of complementary mRNA transcripts. We are employing RNAi for the exploration of cancer-associated pathways including the validation of anti-cancer targets, the potential identification of new targets, the development of novel model systems, and the elucidation/validation of integral components of cancer related phenotypes. To access large-scale synthetic siRNA resources GSS has developed a research collaboration agreement with Qiagen Inc. To date Qiagen has designed and synthesized siRNAs corresponding to approximately 400 genes. To make full use of these resources have developed a high throughput automated synthetic siRNA-lipid "reverse transfection" protocol in a 96 well plate format (BioRobot 8000, Qiagen Inc.). To date we have assayed the knockdown mediated by 267 siRNAs corresponding to 131 human genes. Over 70% of these siRNAs show a statistically significant decrease in the steady state levels of the expression of the gene under study. We are actively investigating the reasons for why some siRNAs fail to silence. In many cases we suspect that a failure to induce silencing often reflects issues related to the expression of different alternative transcripts of the gene in question and single nucleotide polymorphisms that block cleavage by the siRNA-RISC complex. In a limited number of cases we have also investigated the specificity of the siRNAs. In addition to validation we are now expanding this platform to allow us to further our pharmocogenomic studies through the development of a combinatorial library approach.