The Induction of Gene-specific RNAi Against Cancer-associated Genes

基因组规模的分子分析已经彻底改变了实验过程， 影响癌症发生、发展和治疗的基因和途径可以 被识别。这为生物医学研究开辟了许多令人兴奋的途径，但它 也证实了肿瘤发生的遗传和细胞复杂性。的 确定基因沉默机制，RNA干扰（RNAi），最初在 无脊椎动物和后来的哺乳动物细胞，对我们的研究产生了巨大的影响。 了解基因表达的调节和我们调节它的能力 实验性的该项目的重点是假设，分子和表型 RNAi引起的干扰将使人们深入了解癌症的生物学， 抗癌分子靶点。大多数研究的目的是利用RNAi机制， 功能丧失（LOF）分析是蛋白质编码mRNA的基因特异性切割。 利用基于内源性RNA的基因沉默机制（RNA干扰）的技术已经 它迅速发展，用于解剖基因功能关系，并作为一种手段， 进一步的分子目标分析。该项目的总体目标是增强 RNAi技术在癌症生物学研究中的应用。要做到这一点，我们 重点是建立可重复评估影响的方案和测定法， 在分子和功能水平上的RNAi，可用于哺乳动物细胞系模型 适合于肿瘤发生研究的系统。我们正在应用优化和强大的 使用合成的siRNA和其他RNAi效应物在细胞中诱导RNAi的方案，例如 短发夹RNA（shRNAs）和使用定量分析来分析 核糖核酸到目前为止，我们已经研究了siRNAs介导的沉默， ≈250个人类基因和150个shRNA克隆， ≈50个人类基因（后一项研究最初在项目编号 Z01 BC 010614，但该工作现已在本项目工作范围内调整）。这个项目 对于协助和培训CCR研究人员应用 RNAi分析，并建立针对数百个靶向的RNAi筛选方案。 人类基因（参见Z01 BC 010615）。

Genome scale molecular analysis has revolutionized the experimental process by which the genes and pathways that influence the initiation, progression, and treatment of cancer can be identified. This has opened up many exciting avenues of biomedical research, but it has also served to confirm the genetic and cellular complexity that underlies tumorigenesis. The identification of the gene silencing mechanism, RNA interference (RNAi), initially in invertebrates and later in mammalian cells, has had enormous implications for our understanding of the regulation of gene expression and our ability to modulate it experimentally. This project is focused on the hypothesis that molecular and phenotypic perturbations induced by RNAi will give insight into the biology of cancer and identify novel anti-cancer molecular targets. The intention of most studies exploiting the RNAi mechanism for loss of function (LOF) analysis is the gene-specific cleavage of protein encoding mRNAs. Technologies that exploit the endogenous RNA-based gene silencing mechanism, RNAi, have developed rapidly for the dissection of gene-function relationships and as a means of furthering molecular target analysis. The overall goal of this project is the enhanced application of RNAi-based technologies for the study of cancer biology. To do this we are focusing on establishing protocols and assays for the reproducible assessment of the effects of RNAi at a molecular and functional level that can be used in mammalian cell line model systems appropriate for the study of tumorigenesis. We are applying optimized and robust protocols for inducing RNAi in cells using synthetic siRNAs and other RNAi effectors, such as short hairpin RNAs (shRNAs) and the use of quantitative assays for analyzing the efficacy of RNA. To date we have examined the silencing mediated by siRNAs corresponding to over ≈250 human genes and ≈150 shRNA clones corresponding to ≈50 human genes (this latter study was originally detailed under project number Z01 BC 010614 but this work has now been aligned within this project effort). This project continues to be critical for assisting and training CCR Investigators in the application of RNAi analysis, and for the establishment of protocols for RNAi screens targeting hundreds of human genes (see Z01 BC 010615).