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Short hairpin RNA (shRNA) that inhibit gene expression through RNA interference (RNAi) are usually designed through sequence-based rules that undergo continuous revision. An alternative procedure is to convert randomly fragmented cDNA into shRNA templates. The latter procedure increases the number of shRNA sequences per gene and allows one to include genes and splice variants that have not yet been identified. To cover the largest number of target genes and equalize their sequence representation, we have used normalized (uniform-abundance) cDNA of MCF-7 breast carcinoma cells for shRNA library construction. The resulting library is estimated to represent about one half of all the human genes at an average of ~100 shRNA clones per gene. The shRNA library was constructed in an inducible lentiviral vector, allowing for precisely controlled measurement of gene knockdown. RNAi activities were measured by Q-PCR for 101 shRNAs targeting different human genes. We have also constructed an shRNA library from a single gene, firefly luciferase, and measured the activity of 230 luciferase-derived shRNA sequences. Structure-activity analysis of both shRNA datasets has been used to identify structural determinants of shRNA activity. We have used the cDNA-derived shRNA library to select for shRNA sequences that inhibit the growth of MDA-MB 231 cell line, which was previously used to select growth-inhibitory genetic suppressor elements (GSE) by bromodeoxyuridine suicide selection. Cells that passed the selection carried shRNA sequences for a set of genes, some of which were the same as previously identified by GSE selection. shRNA libraries derived from normalized cDNA should provide a useful general tool for function-based gene identification.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA