Screens Using RNAi and cDNA Expression as Surrogates for Genetics in Mammalian Tissue Culture Cells

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Abstract
We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation,and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiasedmanner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinasesin individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treatmammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments havebeen performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, andamino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general typesof screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and geneticinteraction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that canbe scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalianproteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens thatwill allow queries on the roles of all human proteins.