TechnicalreportLarge-scaleexpressionscreeningbyautomatedwhole-mountinsituhybridizationRebeccaQuiringa,BeateWittbrodta,ThorstenHenricha,MiranaRamialisona,CarolaBurgtorfb,HansLehrachb,JoachimWittbrodta,*aDevelopmentalBiologyProgramme,EuropeanMolecularBiologyLaboratory,Meyerhofstrasse1,D-69012Heidelberg,GermanybMax-Planck-Institutfu¨rMolekulareGenetik,Ihnestrasse73,D-14195Berlin,GermanyReceived22December2003;receivedinrevisedform12March2004;accepted31March2004AbstractGeneexpressionprofilingisanimportantcomponentoffunctionalgenomics.Wepresentatimeandcostefficienthigh-throughputwhole-mountinsitutechniquetoperformalarge-scalegeneexpressionanalysisinmedakafish(Oryziaslatipes)embryos.Medakaisamodelsystemideallysuitedforthestudyofmoleculargeneticsofvertebratedevelopment.RandomcDNAclonesfromanarrayedstage20medakaplasmidlibrarywereanalyzedbywhole-mountinsituhybridizationonembryosofthreerepresentativestagesofmedakadevelopment.cDNAinsertswerecolonyPCRamplifiedina384-format.ThePCRproductswereusedtogenerateover2000antisenseRNAdigoxigeninprobesinahigh-throughputprocess.Whole-mountinsituhybridizationwascarriedoutinarobotandabroadrangeofexpressionpatternswasobserved.PartialcDNAsequencesandexpressionpatternsweredocumentedwithBLASTresults,clusteranalysis,imagesanddescriptions,respectively;collectivelythisinformationwasenteredintoaweb-baseddatabase,‘MEPD’(http://www.embl-heidelberg.de/mepd/),thatispubliclyaccessible.q2004ElsevierIrelandLtd.Allrightsreserved.Keywords:Oryziaslatipes;PartialcDNAsequencing;ExpressedSequenceTags;Screening;Automated;Whole-mountinsituhybridization;Geneexpression;High-throughput;Database;Expressionprofile;Embryogenesis;Organogenesis1.IntroductionTheunderstandingofthemolecularmechanismsunder-lyingagivendevelopmentalprocesshasprofitedfromgeneexpressionstudiesbyinsituanalysis.Thisapproachhasbeeninstrumentalintheisolationofalargenumberofnovelgenesthatareexpressedinaspecificspatialandtemporalmanner.Itisacomplementaryapproachtosystematicmutagenesisscreenssinceg...
