title: Organic small hairpin RNAs (OshR): a do-it-yourself platform for transgene-based gene silencing.
abstract: The RNA interference (RNAi) pathway in animal cells can be harnessed to silence gene expression with artificial small interfering RNAs (siRNAs) or transgenes that express small hairpin RNAs (shRNAs). The transgene-expressing shRNA approach has been adapted into large-scale resources for genome-wide loss-of-function screens, whereas focused studies on a narrow set of genes can be achieved by using individual shRNA constructs from these resources. Although current shRNA repositories generally work, they might fail in certain situations and therefore necessitate other alternatives. We detail here a new highly-accessible and rational design of custom shRNAs that utilizes a refined backbone configuration termed the 'organic' shRNA (OshR) platform. The OshR platform is 'organic' because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects. We also demonstrate that the reliability of the OshR platform for gene silencing is increased when sequences target the 3' UnTranslated Region (3'UTR) of a gene. We further compare the OshR platform with the current and emerging shRNA designs, and propose that the OshR platform is a novel approach that can allow investigators to generate custom and effective shRNAs for individual gene functional studies.
authors.affiliation: Department of Biology, Brandeis University, United States; Rosenstiel Basic Medical Science Research Center, United States.
authors.forename: Mei
authors.initials: M
authors.lastname: Zeng
authors.forename: Marissa S
authors.initials: MS
authors.lastname: Kuzirian
authors.forename: Lamia
authors.initials: L
authors.lastname: Harper
authors.forename: Suzanne
authors.initials: S
authors.lastname: Paradis
authors.forename: Takuya
authors.initials: T
authors.lastname: Nakayama
authors.forename: Nelson C
authors.initials: NC
authors.lastname: Lau
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D003001:Cloning, Molecular; D055785:Gene Knockdown Techniques / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis / Q000235:genetics; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D019076:Transgenes; D014981:Xenopus; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; C531201:MIRN451 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D029867:Xenopus Proteins; D014442:Monophenol Monooxygenase; D000072379:NIMA-Related Kinases; C490317:Nek2 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: Gene silencing; RNA interference; shRNA