Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding Evaluation of a Novel, High-Throughput Screening Assay 英文参考文献.docVIP

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Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding Evaluation of a Novel, High-Throughput Screening Assay 英文参考文献.doc

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Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding Evaluation of a Novel, High-Throughput Screening Assay 英文参考文献

EffectsofGeneticMutationsandChemicalExposureson CaenorhabditiselegansFeeding:EvaluationofaNovel, High-ThroughputScreeningAssay WindyA.Boyd1,SandraJ.McBride2,JonathanH.Freedman1,2* 1LaboratoryofMolecularToxicology,NationalToxicologyProgram,NationalInstituteofEnvironmentalHealthSciences,NationalInstitutesofHealth, ResearchTrianglePark,NorthCarolina,UnitedStatesofAmerica,2NicholasSchooloftheEnvironmentandEarthSciences,DukeUniversity,Durham, NorthCarolina,UnitedStatesofAmerica Background.Governmentagencieshavedefinedaneedtoreduce,refineorreplacecurrentmammalian-basedbioassayswith testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive optionbecauseoftheirshortlifecycles,inexpensivemaintenance,andhighdegreeofevolutionaryconservationwithhigher eukaryotes.TheC.eleganspharynxisafavorablemodelforstudyingneuromuscularfunction,andtheeffectsofchemicalson neuromuscularactivity,i.e.,feeding.Currentfeedingmethodologies,however,arelaborintensiveandonlysemi-quantitative. Methodology/PrincipalFindings.Hereahigh-throughputassayisdescribedthatusesflowcytometrytomeasureC.elegans feedingbydeterminingthesizeandintestinalfluorescenceofhundredsofnematodesafterexposuretofluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increasesinfeedinglevelsafterserotoninexposuresweredependentonfoodavailability,whilefeedinglevelsdecreasedin arecoline-exposed nematodesregardlessofthepresenceoffood.Theeffectsoftheenvironmental contaminants,cadmium chlorideandchlorpyrifos,onwild-typeC.elegansfeedingwerethenusedtodemonstrateanapplicationofthefeedingassay. Cadmium exposures above 200mM led to a sharp drop in feeding levels. F