杨文钦(中央研究院生物农业科学研究所筹备处)

2008/3/131楊文欽(中央研究院生物農業科學研究所籌備處)電話:[phone](Office)E-mail:[e-mail]:=wcy實驗藥理學Theuseofmousemodelsforpharmacologicalstudies(模式鼠在藥理學的應用)時間:96/3/21(星期三)早上3-4節(10:10-12:00)及下午5-8節(13:30-17:20)地點:研究大樓4樓458室及各實驗室2008/3/132Mousemodels1.NaturaloccurringmousemodelsNODmice;db/dbmice(leptinreceptormutation)2.Geneticsmodifiedmousemodels2-1.Genetargeting(Knockout,conditionalKO,Knockin):2-2.Transgenesis(TransgenicmiceexpressingWTgene,DNgeneandsiRNA)2-3.ENUmice2-4.NuclearTransfer(Stemcellnucleartransfer(SCNT),Fertilizedeggnucleartransfer)Reference:1.Animalmodels:Jacksonlab()andCharlesRiverLab,Inc.()2.ManipulatingthemouseembryoBrigidHoganetal.3.MousegeneticsandtrangenicsI.J.Jacksonetal4.Journalofbiochemistryandmolecularbiology(2004)37:1215.JournalofImmunology(2007)178:6984-69936.ToxicologyandAppliedPharmacology(2007).219(1).54-61Outlines2008/3/133Geneticsapproaches1.Forwardgenetics(順向遺傳學):為傳統的遺傳學，由(突變株的)性狀去找出控制此性狀之基因的方法。例如ENUmutation,genetrapping,insertionalinactivation.2.Reversegenetics(逆向遺傳學):為現代分子遺傳學，由已知基因序列，再去研究這基因功能的方法。KO,Tg2008/3/134Advantage:1.easybreeding2.shortlifespan3.multiplepulps4.easyhusbandry5.idealmammalianmodel.Use:1.Basicstudiesongeneregulationandfunction2.Studiesonhumandiseaseanditsprogression3.Pharmacological/therapeuticstudies4.DrugdiscoveryandtargetidentificationPropertiesandapplicationsofmiceinmedicalresearch2008/3/1351.GenetargetingKnockout,conditionalKO,Knockin2.Transgenesis-TransgenicmiceexpressingWTgene,DNgeneandsiRNA3.ENUmice4.NuclearTransferStemcellnucleartransfer(SCNT)FertilizedeggnucleartransferHowtoengineermice2008/3/136+Day1:PMSGinjectionDay3:HCGinjectionXXDay4:matingfostermotherWithvasectomizedmales+++Day4:isolationofoocytesandmicroinjectionofDNADNA+Day5:oviducttransfertopseudopregnantfostermotherDay25:birthofoffspringDay46:tailtippingandSouthernblotJacksonetal.MousegeneticsandTransgenicsp232Generationoftransgenicmice(1982)2008/3/137TransgenicvectorpCAS-mCD3-delta-N3flag-LZ6647bpBGHpAN3flagLZmCD3-dalta-enhancermCD3-dalta-promoterNeomycinAmpicillinSV40pASV40Sp6ColE1KpnI(40)NotI(2158)ScaI(6205)XhoI(1071)DraIII(312)BamHI(432)BamHI(2004)EcoRI(426)EcoRI(655)HindIII(926)HindIII(1299)XmaI(1056)XmaI(3292)BglII(1065)BglII(1650)NdeI(25)NdeI(2734)SacI(1827)SacI(2206)SalI(4458)SalI(6646)SpeI(18)SpeI(2727)XbaI(30)XbaI(2739)PstI(1609)PstI(1660)PstI(3534)2008/3/138Transgenicprocedure2008/3/139TransgenicmiceexpressingRNAi(2003)promoterRNAi2008/3/1310EScellsElectroporationG418selectionX+/+Chimeric+/-+/-+/++/+-/-Generationofknockoutmice(1985-1986)KOconstruct2008/3/1311Knockoutconstruct2008/3/1312Homologousrecombination2008/3/1313Conditionalknockout(1992)2008/3/1314CreLoxPsystemFigure1.AmodelofCreFunction.TheloxPsitesrecognizedbyCrearerepresentedwiththickarrowsandtheirDNAsequenceisshownatthebottom.2008/3/1315CreLoxPsystemTheCre-LoxPsystem.TheloxPsitecanbeinsertedoneithersideofapieceofDNA.(A)IfCrerecombinaseisthenexpressedinthecell,theloxPsitewillbecutandjoinedtogether,removingthepieceofDNAbetweenthetwosites.(B)SincetheloxPsiteisdirectional,itcanalsobeusedtoinvertpiecesofDNAwhicharebetweenit.HeretwoloxPsiteswhichareinsertedoneithersideofapieceofDNAinoppositedirections.OnceCreisexpressed,theloxPsitesarebothcutandthepieceofDNAinvertedandreattached.2008/3/1316Cre/loxregulationofgeneexpressionFigure2.Cre/loxRegulationofaProteinExpression.ThegenelacZhasLoxPsequencescontainingastopsignalthatpreventthegenefrombeingexpressed.WhenexposedtotheCreproteintheLoxPandstopsignalareexcisedandthegeneisexpressed.ConditionsinwhichthecreispresentthusregulatedtheexpressionofthelacZgene.2008/3/1317Tissue-specificgeneKOFigure3.Cre/loxMouseBreeding.MicewiththeCreproteinexpressinginaspecificcelltypearebredwithmicethatcontainatargetgenesurroundedbyloxPsites.Whenthemicearebred,thecellscarryingCrewillcausethosecellstolosethetargetgene.2008/3/1318KnockinconstructCell,Vol.97,767–777,June11,19992008/3/1319ENUmice2008/3/1320Stemcellnucleartransfer(SCNT)FertilizedeggnucleartransferNuclearTransfer2008/3/1321NuclearTransfer2008/3/1322Nucleartransfer2008/3/1323UsefulnessofTganimalProducingpharmaceuticals,usefulenzymesandproteinsGenefarmtoproducemeatsBreedingofhigh-qualityanimalsEngineeringgraftedorgansforhumans:hDAFtgScientificresearchesUsefulnessofkoanimalProducingpharmaceuticalsProducingusefulenzymesandproteinsGenefarmtoproducemeatsBreedingofhigh-qualityanimalsEngineeringgraftedorgansforhumansScientificresearchesUsefulnessofnucleartransferProducingexactclones(formedicalresearch,…)ProducingtissuesororganswithidenticalgenesStudiesondiseasesApplicationsofgeneticallymodifiedanimals2008/3/1324Example1EvaluationofBu-Zhong-Yi-Qi-TangontheprotectionagainstListeriaImmunopharmacology(1998)215-1132008/3/1325ListeriainvasionMolecularMedicalMicrobiology(2002)chapter69byMaxSussman2008/3/1326ImmunitytoListeriaMØAct.MØEpitheliaCD8TKillingCD4TMØNKIFNγEpitheliaBloodIntestinesListeria2008/3/1327HOTenhancesListeriaclearanceHOTOralAdm.7daysLMPlatingassay