转基因治病模型.ppt

TransgenicMutationAnimalAssays转基因动物致突变模型,TaoChen,Ph.D.陈涛，博士NationalCenterforToxicologicalResearch,USFDA美国食品药品管理局国家毒理研究中心,Outline概要,IntroductionTransgenicmutationsystemsDevelopmentoftransgenicmutationanimalsProtocolfortransgenicmutationassaysValidationoftransgenicmutationassaysComparisonofmutationsintransgenesandnativegenesTransgenicmutationassaysvs.othergenotoxicassays,andvs.carcinogenicityassaysUseoftransgenicanimalmutationassaysRegulatoryuseforinvivogenotoxicitytestingMechanisticstudiesConclusion,Introduction导言,WhatisMutation?什么是突变？,MutationsareheritablechangesinthenucleotidesequenceofDNAMutationsinsomaticcellscancauseneoplasmsandpossiblyagingMutationsingermcellsareresponsibleforalargenumberofgeneticdiseases,includingbirthdefectsandcancer,MutationandCancer突变和癌症,MutationisnotequaltocancerCancer:adiseasecausedbysomaticcellmutations,Mutationasabiomarkerofcancer突变可用作癌症的生物指标,Mutationassaysasshort-termtestsforcarcinogenicityInvitro:Salmonellamutationassaysandgenemutationsinculturedmammaliancells.Invivo:Hprtassay,Aprtassay,TkassayandtransgenicassayInvivoevidenceismoreimportantthaninvitroevidence,Transgenicmutationsystems转基因致突变系统,Transgenicanimalsformutationassays检测突变的转基因动物,Transgenicanimalsareanimalswhosecellscontainforeigntargetgenes(reportergenes)reportergenesarenormallylocatedonshuttlevectorsthatarederivativesofbacteriophageorplasmidsThevectorsrecoverthemutationaltargetfromrodentsformutantdetectioninbacteria,Shuttlevector穿梭载体,Ashuttlevectorcontainingreportergene(s)isconstructedusingrecombinantDNAtechnologies.FollowingisaLIZshuttlevectorusedforBigBluetransgenicrodents,Transgenicrodentmutationmodels转基因动物突变模型,BigBlueRats大蓝大鼠,Procedurefortransgenicrodentmutationassays转基因动物致突变实验步骤,Treatment药剂处理,Forregulatorysafetyassessment,theInternationalWorkshoponGenotoxicityTestProcedures(IWGT)recommendsrepeattreatmentofanimalsfor28dayswiththemaximumtolerateddose(MTD)Twofurtherdosegroupsreceiving1/3and2/3theMTDaredesirableincasethatthenumberofanimalsinthehighdosegroupisreducedbyagenttoxicityIftherearenosignificantdifferencesbetweenthesexesintermsoftoxicityormetabolism,theIWGTrecommendsthatmaleanimalsshouldnormallybeused,Mutantmanifestationtime突变体显示时间,MutantmanifestationtimeistheperiodbetweenthelasttreatmentandthetimeofsamplingtissuesformutationThemanifestationtimeisdependentonthetissuethatisassayedtheIWGTrecommendsageneralscheduleofsampling3daysafter28daysofrepeatedtreatmentforalltissues,Collectionoftissues组织收集,AllpotentialtargettissuesfromwhichreasonableamountsofDNAcanberecoveredshouldbecollectedTominimizetheinsitudegradationoftheDNA,eachtissueshouldbedissected,wrapped,andflash-frozeninliquidnitrogenStorethefrozentissuesat80C,IsolationofgenomicDNA分离染色体组DNA,GenomicDNAshouldbe100300kborlargerforefficientphagerescueTheRecoverEaseDNAisolationkit(Stratagene)isrecommendedfortheisolationofhighmoleculargenomicDNAGenomicDNAcanbestoredinaTEbufferat40C,protectedfromlight,foruptoonemonth,Statisticsandcriteria统计和准则,200,000transgenesperanimalfromatleast5animalspertreatmentmustbescreenedtodetecta2-foldincreaseintheMFIfanappropriatestatisticalanalysisisperformed,thedifferencebetweentheMFsfromthetreatedandcontrolgroupscanbeusedtojudgeatestingagentpositiveornegative,ValidationofTransgenicSystems转基因系统的确证,Dothetestsystemsmeasurewhatweareintendingtomeasure?Aretheassayspredictiveoftheendpointsofinterest?,MFsinLymphocyteHprtandlacIvs.LiverlacIinN-OH-AAFTreatedRatsN-OH-AAF引诱的淋巴细胞Hprt和lacI基因突变频率以及肝基因突变频率,MF(x10-6),Hprtvs.lacIMFInducedbyThiotepainLymphocytesThiotepa引诱的淋巴细胞Hprt和lacI基因突变频率,MF(x10-6),ComparisonsofmutationsintheHprtandlacIgenesHprt和lacI基因突变的比较,Transgenicmousemutationassayvs.mousebonemarrowmicronucleustestforpredictingmousecarcinogenicity小鼠致癌性的预测：转基因小鼠致突变测试对小鼠骨髓微核体测试,Summaryonresultsintransgenicrodentassaysvs.genotoxicityinvitrotests致突变结果比较：转基因动物致突变测试对体外遗传毒理测试,Advantagesanddisadvantages优点和缺点,Mainadvantage:MutationscanbedetectedinanyorgansotargetorgansofcarcinogenscanbeevaluatedMaindisadvantage:Detectprimarilypointmutationandlarge-scalegenealterationswillbedetectedwithlowefficiency,Useoftransgenicanimalmutationassays转基因动物致突变模型的应用,Useforregulatorypurposes应用于规章的制定和法规的执行,TheneutralityofthetransgenesallowstheaccumulationandpersistenceofmutationsinvivoanditmaketheselectionofexpressiontimeseasierTheresultsfromtransgenicmutationassaysarereproducibleandtheassaysaretransferablebetweendifferentlaboratorieslowercostsandtheuseoffeweranimals,Fillagapinregulatorytestingstrategies添补检测系统中的不足之处,Currentinvivogenotoxicityassaysthatarerecommendedbyregulatoryagenciesarethechromosomalaberration,micronucleusandUDSassaysReplaceUDSassayComplementeachotherwithchromosomalaberrationandmicronucleusassaysinsafetyassessments,Promotionbyregulatoryagencies执法机构的促进推广,WHOispreparinganEnvironmentalHealthCriteriadocumentontransgenicrodentmutationassaysandtheiruseintoxicityassessmentTheOrganizationforEconomicCo-operationandDevelopment(OECD)aredevelopingguidelinesforuseoftransgenicrodentmutationassaysManyregulatoryagenciesrecommenduseoftransgenicmutationassays,Mutationtypes突变类型,Aristolochicacids（AAs）-inducedtotalDNAadducts马兜铃酸的DNA加合物,KidneyLiver,AAs-inducedcIImutantfrequency(MFs)马兜铃酸诱导的突变频率,KidneyLiver,AutoradiographicprofilesofDNAadductsusing32P-postlabelingassay,spot1:dA-AAIspot2:dG-AAIspot3:dA-AAII,Kidney,Liver,ControlAA(10mg/kg),100,83,100,41,Total,0,0,0,0,Typeofmutation,0,0,2,1,Tandem-base,1,1,20,8,Frameshift,17,14,2,1,A:TG:C,5,4,2,1,A:TC:G,50,42,2,1,A:TT:A,4,3,25,10,G:CT:A,16,13,32,13,G:CA:T,7,6,15,6,G:CC:G,%,Number,%,Number,AAs(10mg/kg),Control,SummaryoftheindependentmutationsintheliverandkineycIIgenefromtheAAs-treatedandcontrolBigBluerats,100,125,100,55,4,5,2,1,2,2,0,0,2,3,15,8,9,11,5,3,1,1,5,3,54,68,5,3,7,9,9,5,16,20,55,30,5,6,4,2,%,Number,%,Number,Complex,AAs(10mg/kg),Kidney,Control,Liver,P=0.106,P=0.065,Conclusio