Functional Selection of shRNA Loops from Randomized Retroviral Libraries 英文参考文献.docVIP

FunctionalSelectionofshRNALoopsfromRandomized RetroviralLibraries StigM?lgaardRaskJensen1,AlexanderSchmitz1,FinnSkouPedersen1,J?rgenKjems1,2* ,Jesper BertramBramsen1,2 1Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 2Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark Abstract Gene silencing by RNA interference (RNAi) can be achieved by the ectopic expression of tailored short hairpin RNAs (shRNAs) which after export to the cytoplasm are processed by Dicer and incorporated into the RNA induced silencing complex(RISC). Design rulesfor shRNAs havebeenthe focusofseveral studies,butonlyafewreports haveturnedthe attentiontothesequenceoftheloop-region.Inthisworkweselectedhigh-functionalandlow-functionalshRNAloopsfrom retroviral hairpin-loop-libraries in an RNAi reporter assay. The procedure revealed a very significant and stem sequence- dependent effect of the loop on shRNA function and although neither strong consensus loop sequence nor structural motifscouldbeidentified,apreferredloopsequence(59-UGUGCUU-39)wasfoundtosupportrobustknockdownwithlittle stemsequencedependency.ThesefindingswillserveasaguidefordesigningshRNAswithimprovedknockdowncapacity. Citation: Jensen SMR, Schmitz A, Pedersen FS, Kjems J, Bramsen JB (2012) Functional Selection of shRNA Loops from Randomized Retroviral Libraries. PLoS ONE7(8):e43095.doi:10.1371/journal.pone.0043095 Editor:JohnJ.Rossi,BeckmanResearchInstituteoftheCityofHope,UnitedStatesofAmerica ReceivedFebruary3,2011;AcceptedJuly18,2012;PublishedAugust17,2012 Copyright: ? 2012 Jensen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwassupportedbytheRIGHTEUconsortium(no.LSHB-CT-2004-005276),theSIROCCOEUconsortium(LSHG-CT-2006-037900),theDanish TechnicalResearchCouncil,theDanishStra