Protein kinases are key regulators of all aspects of neoplasia, including proliferation, angiogenesis, invasion, and metastasis. The completion of human genome allowed a more comprehensive examination of the entire kinase gene family that was last suggested to have 518 genes. These proteins mediate most of the cellular signal transduction processes in eukaryotic cells. By modification of substrate activities, they control transcription, metabolism, cell movement, cell proliferation, differentiation, and apoptosis. In addition to their critical roles in cancer development, protein kinases are also implicated in wide range of human diseases in metabolic system, immune system and central nervous system. Protein kinases also emerge as great targets for drug discovery and development, particularly in the area of cancer. Kinases are often critical in regulating signal transduction in response to external signals. They are enzymes that are good targets for traditional pharmaceutical drug development, particularly for screening chemical inhibitors. Kinase inhibitors can be selective due to structure differences in the active sites. Furthermore, as many receptor tyrosine kinases are critical for cancer development, they are also good targets for antibody therapies. With the success of Glivec, Iressa, Herceptin, and Erbitux, more kinase specific drug candidates are in the race to reach the patients. At OriGene, we are committed to facilitate the research and drug development in the arena of protein kinases by generating critical reagents, both expression cDNA clones and inhibitory clones for the entire family of protein kinases. OriGene has built a collection of 460 human full-length kinase genes in an expression system. In the proposed project, we will develop a complete set of validated RNA interference (RNAi) expression vectors for the entire family of human protein kinases. To reach this end, we adapted and optimized a robust shRNA (small hairpin RNA) expression library approach where the gene specific shRNA vectors are generated directly from the cDNA of kinase genes. With this kinase shRNA library method, one can generate a large number of shRNA expression vectors from their cDNA in a throughput manner. The method is faster, cheaper and better than the commonly used synthetic oligos-based plasmid construction approach. The overall objectives of this Phase I grant are: 1) to generate kinase shRNA expression libraries utilizing the available kinase genes; 2) to sequence validate about 8 unique shRNA vectors per kinase gene; 3) to functionally validate at least one shRNA vector per kinase gene in inhibiting the target gene expression. We believe that these products will be essential for all cancer researchers by allowing long-term studies of kinase knockdown both in vitro and in vivo. Phase II of this project is to develop and validate shRNA expression vectors for 5,000 human genes that are implicated in human health and medicine
