Generation of a potent kinase siRNA library using Dicer

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.