A random shRNA-encoding library for phenotypic selection and hit-optimization.
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.
Wang
Yongping
Y
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Wang
Yun E
YE
Cotticelli
M Grazia
MG
Wilson
Robert B
RB
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D016296:Mutagenesis; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012190:Retroviridae / Q000378:metabolism
D007377:Interleukin-3; D035683:MicroRNAs; D034741:RNA, Small Interfering; D053148:Caspase 3
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