a random shrna-encoding library for phenotypic selection and hit-optimization一个随机shrna-encoding表型选择和hit-optimization图书馆.pdfVIP

A Random shRNA-Encoding Library for Phenotypic Selection and Hit-Optimization 1 2¤ 1 1 Yongping Wang , Yun E. Wang , M. Grazia Cotticelli , Robert B. Wilson * 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 School of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America Abstract RNA interference (RNAi) is a mechanism for inhibiting gene expression through the action of small, non-coding RNAs. Most existing RNAi libraries target single genes through canonical pathways. Endogenous microRNAs (miRNAs), however, often target multiple genes and can act through non-canonical pathways, including pathways that activate gene expression. To interrogate all possible functions, we designed, synthesized, and validated the first shRNA-encoding library that is completely random at the nucleotide level. Screening in an IL3-dependent cell line, FL5.12, yielded shRNA-encoding sequences that double cell survival upon IL3 withdrawal. Using random mutagenesis and re-screening under more stringent IL3-starvation conditions, we hit-optimized one of the sequences; a specific nucleotide change and the creation of a mismatch between the two halves of the stem both contributed to the improved potency. Our library allows unbiased selection and optimization of shRNA-encoding sequences that confer phenotypes of interest, and could be used for the development of therapeutics and tools in many fields of biology. Citation: Wang Y, Wang YE, Cotticelli MG, Wilson RB (2008) A Random shRNA-Encoding Library for Phenotypic Selection and Hit-Optimization. PLoS ONE 3(9): e3171.