基因敲除模式小鼠的饲养与交配

基因敲除模式小鼠的饲养与交配Painforlaboratorymicerevealedin'human'facialexpressionsWhatareweusuallyusing?GeneticallyEngineeredMouseModelsTransgenicKnockoutKnockinResourceGiftMadebyyourownMadebyBiocytogenGeneralprinciplesforbreedingFundamentalsofMouseReproductionSexualmaturity:6~8weeksReproductivelifespan:7~8monthsFertilityGestation:18~21daysGenerationtime:12weeksLittersize:2~12pups/litterWeaningage:18~28daysBreedingPerformanceFactorsHybridvigor---more,larger,andhealthierlittersthaninbredstrains.Strain-specificbehaviors---AggressivebehaviorsorpoormothersMutationsandtransgeneeffects---Embryoniclethal,orinfertilityorreducedfertilityOptimizingBreedingPerformanceReplacebreedersregularlyReplacenon-productivebreeders~60daysMatemiceearlyUseexperiencedmalesKeepaccuratebreedingrecordsOptimizingBreedingPerformanceLitterfosteringDeterminingpregnancyMatingnumerousfemalessimultaneouslyManagingSmallColoniesMaintainaminimumofsixbreedingpairsKeeptheagerangeofyourbreedersbetweentwoandeightmonthsoldRetaintwogenerationsofastrain,anddonoteliminateoneuntilthenextoneisproducingMonitorbreedingperformancecloselyConsidercryopreservingastrainBreedingSchemesWhatgenotypesdoIneed?mostefficientwaytoproducethem?Willappropriatecontrolsbeavailable?geneticbackgroundHowaregenotypesidentified?phenotype?molecularassay(e.g.PCR)?WhataboutLinkage?X-linkedormultiplegenes?Reproductiveissues(sterility)Phenotypeissues(lethality)ChoosingaBreedingSchemeSimpleBreedingSchemes+/-Heterozygous+/+Wildtype+/-Heterozygous+/+Wildtype50%50%+/-wildtypeKnockoutSimpleBreedingSchemes+/-Heterozygous+/-Heterozygous+/-Heterozygous+/+Wildtype50%25%-/-Homozygous25%SimpleBreedingSchemes+/-Heterozygous-/-Homozygous+/-Heterozygous-/-Homozygous50%50%BackcrossingbreedingN1N#=backcrossgenerationnumberF#=brotherXsistergenerationnumberComplexBreedingSchemesTransgenicmice•Foundermice:transgenicmicethatdevelopfromtheinjectedeggs•Transgeniclines:derivedfromthesefoundersTransgenicmiceBreedingSchemesfortransgenicmiceTg/+Heterozygous+/+WildtypeTg/+Heterozygous+/+Wildtype50%50%GenotypingstrategyTg-FTg-R---InternalPositiveControlControls:wildtypelittermatesBreedingSchemesfortransgenicmiceGenotypingstrategyTg-FTg-R---InternalPositiveControlControls:wildtypelittermates1.QPCR2.progenytestingComplexBreedingSchemesGermlineKnockoutmiceGermlineKnockoutmiceGenotypingstrategyGenotypeWT-F/RWT-F/Neo-R－/－-+＋/－++＋/＋+-Controls:WildtypeorHeterozygousComplexBreedingSchemesConditionalknockoutmiceGeneralbreedingschemeFrtFrtLoxPLoxPE1E2E3E4E1E2E3E4E1E3E4MatewithFlp-deletermouseMatewithTissuespecificCremouseTargetedallele(fl-neo)Floxallele(fl)Deletedallele(Δ)loxP-FloxP-RE1E2E3E4Wildtypeallele(WT)WT-FWT-RFrt-FFrt-R3’loxP-F3’loxP-RNeoremovalGenotypeWT-F/RFrt-F/Frt-RloxP-F/loxP-RFlp-deleterfl/+,Flp/+++++GenotypingstrategyGenotypeWT-F/RFrt-F/Frt-RloxP-F/loxP-RFlp-deleterCrefl/+,Cre/++++-++/+,Cre/+++--+fl/+,+/++++--+/+,+/+;++---Crossingwithtissue-specificCremouseGenotypingstrategyControls:+/+,cre/+;fl/+,+/+;+/+,+/+;Conditionaldeletionfl/+,Cre/+fl/+,Cre/+Xfl/+,Cre/+fl/+,Cre/+XXfl/+，+/+fl/fl,+/+fl/fl,Cre/+fl/+,Cre/++/+,Cre/+fl/+,+/+fl/fl,+/+GenotypeWT-F/RloxP-F/loxP-RCrefl/fl,Cre/+-++fl/+,Cre/+++++/+,Cre/++-+fl/+,+/+++-fl/fl,+/+-+-GenotypingstrategyControls:+/+,cre/+;fl/fl,+/+;+/+,+/+;ConditionalconverttoGermlineKnockoutE1E3E4MatewithCre-deletermouseTargetedallele(fl-neo)Deletedallele(Δ)loxP-FloxP-RE1E2E3E4Wildtypeallele(WT)WT-FWT-R3’loxP-F3’loxP-RCre/CreorCre/+GenotypeWT-F/R3’loxP-F/3’loxP-RCre-deleterΔ/+,Cre/+++++/+,Cre/++-+ConditionalconverttoGermlineKnockoutGenotypingstrategyControls:+/+,Cre/+ConditionalconverttoGermlineKnockoutΔ/+Cre/+Δ/+Cre/+Δ/ΔΔ/++/+GenotypeWT-F/R3’loxP-F/3’loxP-RCre-deleterΔ/Δ-+-Δ/+++-+/++--GenotypingstrategyControls:Δ/+;+/+Long-TermColonyMaintenanceReplacebreedersfromatrustedvendoreach5to10generationsEstablishageneticqualitycontrolplanGenomescans-SNPs,MITs,andbiochemicalmarkers,removespontaneousmutantsCryopreserveuniquestrainsLong-TermColonyMaintenanceDisasterscomeinmanyformsForcolony“sleep”Forinsuranceandpeaceofmind….developmentandbasicphenotypingofaGMmousecantake2-3yearsandcostmorethan￥500,000CryopreservationCryopreservationoptionsReducecost