Acute Drug Treatment in the Early C. elegans Embryo 英文参考文献.docVIP

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Acute Drug Treatment in the Early C. elegans Embryo 英文参考文献.doc

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Acute Drug Treatment in the Early C. elegans Embryo 英文参考文献

AcuteDrugTreatmentintheEarlyC.elegansEmbryo AnaCarvalho1.,SaraK.Olson1.,EdgarGutierrez2.,KellyZhang1,LisaB.Noble1,EstherZanin1 ,Arshad Desai1,AlexGroisman2*,KarenOegema1* 1LudwigInstituteforCancerResearch,DepartmentofCellularandMolecularMedicine,UniversityofCaliforniaSanDiego,LaJolla,California,UnitedStatesofAmerica, 2DepartmentofPhysics,UniversityofCaliforniaSanDiego,LaJolla,California,UnitedStatesofAmerica Abstract Geneticandgenome-wideRNAiapproachesavailableinC.elegans,combinedwithtoolsforvisualizingsubcellularevents with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functionalgenomicanalysisofcellularprocesses.However,alimitationofthissystemhasbeentheimpermeabilityofthe embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilizeandimmobilizeembryosforacuteinhibitortreatmentinconjunctionwithliveimaging.Toidentifyameansto permeabilizetheeggshell,weusedadyeuptakeassaytoscreenasetof310candidategenesdefinedbyacombinationof bioinformaticcriteria.Thisscreenidentified20geneswhoseinhibitionresultedin.75%eggshellpermeability,and3that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mountpermeabilizedembryosforacutedrugadditioninconjunctionwithliveimaging,wecombinedoptimizedinhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developmentsenablethetemporallycontrolledintroductionofinhibitorsformechanisticstudies.Thismethodshouldalso open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-widephenotypicdatasets. Citation: Carvalho A, Olson SK, Gutierrez E, Zhang K, Noble LB, et al. (2011) Acute Drug Treatment in the Early C. elegans Embryo. PLoS ONE 6(9): e24656. doi:10.1371/journal.pone.0024656 Editor:BenLehner,CentreforGenomicRegulation,Spain Re