转基因动物致突变模型

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 Followingisa LIZshuttlevectorusedforBigBluetransgenicrodents 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 frozeninliquidnitrogenStorethefrozentissuesat 80 C IsolationofgenomicDNA分离染色体组DNA GenomicDNAshouldbe100 300kborlargerforefficientphagerescueTheRecoverEaseDNAisolationkit 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 T G C 5 4 2 1 A T C G 50 42 2 1 A T T A 4 3 25 10 G C T A 16 13 32 13 G C A T 7 6 15 6 G C C 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 Conclus