The proposed research will enable mammalian cells to be used in mutation/selection experiments to identify genes involved in cellular processes. The objective of the proposal is to develop procedures for preparing siRNA libraries using genomic DNA and cDNA samples as the source material for siRNA templates. Libraries of siRNA vectors could comprise 1,000,000,000 to 1,000,000,000,000 unique siRNA expression vectors, making it possible to cover entire mammalian genomes with overlapping siRNAs. Mammalian cell populations transfected or transduced with siRNA vector libraries will be placed under selection to create cell populations expressing a desirable phenotype. The siRNA vector(s) integrated in the genomes of the selected cells will be amplified, cloned, and sequenced to reveal the siRNA template sequence. The siRNA sequences will be used to identify the genes whose down-regulation create the phenotype that was selected. The siRNA vector libraries should enable mutation/selection experiments to be used to correlate gene function and identity in mammalian cells.
