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otherkCre‐loxTechnologyandCrelox Technology and Breeding Schemes in Mouse Models for Research Andy Schile, PhDTechnical Information ServicesotherkNotable ContributionsGeorge Snell, PhD & Nobel PrizeRecipientg,pDiscoveries of immune system function formed the foundation for tissue and organ transplantationLeroy Stevens, PhDLaid the foundation for modern embryonic ElibthRllPhDystem cell researchElizabeth Russell, PhDPioneered the use of bone marrow transplantsotherkTheJacksonLaboratoryThe Jackson LaboratoryRhiiiidResearch: investigating genetics and biology of human diseaseResources: JAX®Mice & Services, bioinformatics data, technical publications and more…Education:world‐class courses, internships and other programsotherkJAX® MiceJAXMiceThe Gold Standard for Biomedical Research•NIH funded resource •4,500 strains and growing ,gg–2.7 million mice shipped annually•Unsurpassed genetic quality & animal health•Best characterized & referenced ~100 new pubs/week•Common inbred strains (C57BL/6J, BALB/cJ, DBA/2J) support development/collectionofspecialtystrainsandothervaluabledevelopment/collection of specialty strains and other valuable community research resources otherkOnlineResourcesOnline Resources®b•JAX®Mice Database•Mouse Genome Informatics•CrePortal•Mouse Phenome Database –Most Characterized‐cgi/phenome/mpdcgi?rtn=strains/details&stocknum=000664otherkStrain SBaselineSurveysBaseline Blood PressureotherkJAX® Services•Breeding & colony management•Revolutionary cryopreservation & recovery•Phenotyping & efficacy testing•Genetic research services•Surgical & preconditioning servicesUfitithlidttfthtit•Use of innovative technologies and state‐of‐the‐art equipment•Flexibility to develop customized approaches JAX® Mice & Servicesjaxservices@jax.org•1-800-422-6423•1-207-288-5845• OverviewBiClhi•Basic Cre‐lox mechanism•Strain types and breeding schemes –General knockouts–Inducible knockouts–Tissue‐specific knockouts–Reporters•Cre‐lox web resources (finding mice, Cre activity data, and more)otherkARevolutionaryGeneticToolA Revolutionary Genetic ToolCltBacteriophageCre‐lox system•Natural part of P1 bacteriophage viral life cycley•Viral DNA injected into bacteria, circularized using Cre‐lox,and replicatedfordevelopmentofnewreplicated for development of new virusesCreotherkCre‐lox Successfully Engineered inOtherOrganismsin Other OrganismsY•Yeast•Plantsllll•Mammalian cell cultures•MiceAllows•Alteration & deletion of DNA•Regulation of location and timing of gene expressionotherkA Simple, Two Component SystemCre recombinaseSitifitlbitiCre•Site‐specific enzyme, catalyzes recombination between two loxPsitesloxPsiteloxPsite•34 base pair DNA sequence•Locationandorientationdeterminesrecombinationresult:•Location and orientation determines recombination result:–Deletion–InversionATAACTTCGTATAGCATACATTATACGAAGTTAT–TranslocationAbundant possibilities for genome manipulation!Reviewed inNagy A. 2000. Genesis 26(2):99‐109. PMID: 10686599otherkCre ‐lox DeletionFloxed target geneloxPloxPGeneXCreexcisionXGeneXloxPCreexcision GeneXKnockout alleleloxPotherkCre ‐lox InversionloxPloxPGeneXCreloxPloxPGeneXloxPotherkCre ‐lox TranslocationChr 3loxPChr 6loxPCreCre loxPReciprocal Translocation (3;6)loxPotherkCre ‐lox Knockout Breeding SchemegEIICxEIIaCreloxPGeneXloxPloxPGeneXloxPGeneXGeneXloxPmouse ‐floxed gene/alleleCremouse –cretransgene (Tg) widespread expression promoterFVB/N‐Tg(EIIa‐Cre)C5379Lmgd/J(Stock # 003314)Offspring:50%heterozygousknockoutafter1generationGeneXOffspring: 50% heterozygous knockout after 1 generationotherkCre ‐lox Knockout Breeding SchemegGeneXGeneXXOffspring 2ndgeneration: 25% homozygous knockoutOspggeeato5%ooygousocoutotherkInducible CreMouse ModelsxloxPmouse ‐floxed gene/alleleInducibleCremouse –tamoxifen dependent Cre functionEx:B6.Cg‐Tg(Cre/Esr1)5Amc/JEx: B6.CgTg(Cre/Esr1)5Amc/J(Stock # 004682)if2 Generations+ TamoxifenloxPGeneXloxPInducehomozygousknockoutofGeneXwithtamoxifenloxPGeneXloxPInduce homozygous knockout of GeneX with tamoxifenotherkCre ‐lox Tissue‐SpecificKnockoutpfxxlfAlbCreloxPmouse ‐floxed gene/allele2 GenerationsLiver‐specific cretransgeneEx: B6.Cg‐Tg(Alb‐Cre)21Mgn/J(Stock # 003574)AlbCreloxPGeneXloxPloxPGeneXloxPliverotherkImproving Conditional Knockout EfficiencyAlbcreGeneXxGeneXloxPGeneXloxPGeneXTraditional knockoutConditional cre‐lox knockoutloxPGeneXloxPAlbcreotherkCre ‐lox Disease ModelsB6.129S4‐Krastm4Tyj/J (Stock # 008179)PromoterloxPloxPKras G12D oncogeneSTOPXXPromoterloxPKras G12D oncogeneotherkCre ‐lox Disease ModelsB6.129S4‐Krastm4Tyj/J (Stock # 008179)xMMTVCreloxPSTOPKras G12DloxPSTOCK Tg(MMTV‐cre)4Mam/J(Stock#003553)(Stock # 003553)loxPKras G12DOnly one round of breeding neededotherkCre ConsiderationsGermlinedeletion•2‐3 generations of breeding•Cre not required (& bred out) aftergermlinede