New shRNA vectors for mammalian genetic analysis

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BACKGROUND. Genetic analysis in model organisms including yeast, C. elegans and fruit flies have yielded many fundamental discoveries in basic biology that ultimately resulted in breakthroughs in our understanding of the disease mechanisms of cancer. The lack of genetic tools in mammalian systems has significantly hampered genetics studies in human cells. The advent of RNAi technology has made it possible to conduct such analysis. PURPOSE. We aim to develop new mammalian vector systems that express shRNAs and cDNAs efficiently. This will enable us to rapidly introduce defined genetic changes into cells to create the desired genotype. SIGNIFICANT MATERIALS AND METHODS. Expression vectors for shRNAs and cDNAs that enable the rapid immortalization of primary human cells and the introduction of multiple oncogenic lesions. FY2013 ACCOMPLISHMENT. In 2012, we established a vector system and validated shRNAs against tumor suppressor genes. In 2013, we have constructed cell lines with defined oncogenic perturbations, including KRAS mutations, to analyze the functional consequences of these alterations.