Generation of a potent kinase siRNA library using Dicer

使用 Dicer 生成有效的激酶 siRNA 文库

• 批准号: 6933695
• 负责人: ZAIREN SUN
• 金额: $ 16.44万
• 依托单位: ORIGENE TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933695
• 关键词: RNA interference; double stranded RNA; gene induction /repression; genetic library; genetic screening; molecular cloning; nucleic acid chemical synthesis; nucleic acid metabolism; nucleic acid structure; protein kinase; ribonuclease III; small interfering RNA; technology /technique development

DESCRIPTION (provided by applicant): siRNA has emerged as a powerful genomics tool for functional analysis of human genes and potential drug discovery. This technology holds great promise for genome-wide functional genetic screens in mammalian cells. However, such screens have been hampered by a lack of effective siRNA product to achieve a highlevel knockdown on a large scale. Conventional siRNA construction methods such as chemical synthesis and short hairpin RNA (shRNA) expression vectors require testing of at least four siRNA oligos or shRNA constructs, and the gene-silencing efficacy is only around 75%. Currently, computer algorithms do not exist to predict a siRNA's effectiveness. The best approach to improve these shortcomings in designing siRNAs and shRNAs for RNAi studies is to mimic the natural way that RNAi machinery target and destroy specific cellular and viral RNAs. This can be accomplished by synthesizing long double-stranded RNAs followed by cleavage with the Dicer enzyme. This would improve the efficacy to 100%. As a provider of genomics tools, OriGene Technologies, Inc. has released 22,000 unique full-length genes to the research community. In addition to the full-length clones, we have also isolated and sequenced 800,000 individual clones from our primary cDNA libraries. The availability of such a large pool of cDNA clones provides a unique resource for the development of derivative products such as for the generation of siRNA libraries when the clones are used in conjunction with Dicer. Phase I of this project aims to optimize experiments to synthesize siRNA using Dicer, and compare simultaneously the gene-silencing effects with multiple siRNAs and shRNAs constructs designed for 24 specific kinase genes. Phase II of the project will focus on the production of siRNAs for the complete kinase gene family and real-time PCR validation of gene-silencing by cotransfection of full-length kinase clones and corresponding Dicer siRNAs. We believe that our technology platform is reliable and innovative, and the outcome of this project will be assuredly a commercial success.

描述（由申请人提供）：siRNA已成为用于人类基因功能分析和潜在药物发现的强大基因组学工具。这项技术为哺乳动物细胞的全基因组功能遗传筛选带来了巨大的希望。然而，这种筛选受到缺乏有效siRNA产物的阻碍，无法实现大规模的高水平敲低。传统的siRNA构建方法如化学合成和短发夹RNA（shRNA）表达载体需要测试至少四种siRNA寡核苷酸或shRNA构建体，基因沉默效率仅为75%左右。目前，还没有计算机算法来预测siRNA的有效性。在设计用于RNAi研究的siRNA和shRNA时，改善这些缺点的最佳方法是模拟RNAi机制靶向和破坏特定细胞和病毒RNA的自然方式。这可以通过合成长的双链RNA，然后用Dicer酶切割来实现。这将使效率提高到100%。作为基因组学工具的提供商，OriGene Technologies，Inc.已经向研究界公布了22,000个独特的全长基因。除了全长克隆外，我们还从我们的初级cDNA文库中分离并测序了80万个单独的克隆。如此大的cDNA克隆库的可用性为衍生产品的开发提供了独特的资源，例如当克隆与Dicer结合使用时，用于产生siRNA文库。本项目的第一阶段旨在优化使用Dicer合成siRNA的实验，并同时比较针对24个特定激酶基因设计的多种siRNA和shRNA构建体的基因沉默效果。该项目的第二阶段将专注于生产完整激酶基因家族的siRNA，并通过共转染全长激酶克隆和相应的Dicer siRNA进行基因沉默的实时PCR验证。我们相信我们的技术平台是可靠且创新的，该项目的结果肯定会取得商业成功。