Cre Lox Breeding for Beginners, Part 1 | JAX Mice and Clinical ...

The Cre/lox system is one of the most powerful and versatile tools developed for mouse genetics. It gives mouse researchers sophisticated ... 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MiceinBiomedicalResearch AreasofResearch Addiction Aging Alzheimer's&OtherDementias Cancer Diabetes Microbiome Rarediseases Vision ClinicalKnowledgebase MaineCancerGenomicsInitiative News  JAXNews JAXBlog MinutetoUnderstanding Subscribe PressRoom TheSearchMagazine ExplorebyTopic AboutUs  FastFacts OurPeople&Culture OurValues Diversity,Equity&Inclusion FacesofJAX OurLeaders WorkWithUs OurCampuses&Communities OurImpact CollaborationatJAX OurMedicalImpact OurEconomicImpact OurCommunityImpact OurEnvironmentalImpact OurHistory Careers ContactUs Give JAXHome NewsandInsights JAXBlog CreLoxBreedingforBeginners,Part1 ByPeterKelmenson BlogPostSeptember23,2011 Simplyput,theCre/lox systemrocks.Itisoneofthemostpowerfultoolsinthemousegeneticist’stoolbox.Why?Well,itenablesthemtogeneratetissue-specificandinducibleknockoutsandtherebyhaveexquisitecontroloverthelocationandtimingofgeneexpression–importantstuffwhendeletingacertaingeneeverywhereorduringdevelopmentleadstoanembryoniclethalphenotype.And,itcanbeusedtoturntransgeneexpressiononoroff,trackindividualcellsorcelllineages(asinthe Brainbow or Confetti mice),generate inversions or translocations,andreportgeneexpression.MorenovelandsophisticatedusesfortheCre/lox systemarebeingdevelopedallthetime. ResearchmousemodelsthatincorporatetheCre/lox systemtypicallyinvolvegeneratingdouble, triple,quadruple,oreven n-tuple (isthatreallyaword?)mutant/transgenicmice.Asyoucanimagine,breedingsuchmicecangetaweebitcomplicated.Our TechnicalInformationServices(TIS) isreceivingmoreandmorerequestsforhelpindesigningbreedingschemesforproducingCre/lox mice.Explainingtheseschemescanbedifficult,andIswearthatIcansometimesfeelcustomers’eyescrosswhileI’mtryingtoexplainthemoverthetelephone.So,inthispost,I’moutliningatypicalbreedingschemethatIhopewillhelpdemystifytheprocessofusingtheCre/lox systemtogeneratetissue-specificorinducibleknockoutmice.(Inafuturepost,Iwilltacklethebreedingschemesforusingthesystemtoturntransgenesonoroff.IwillalsoexplainhowtouseCrereporters). Knockouts:remember,onecrossjustisn’tenough! HereisthemostefficientbreedingschemeforgeneratingCre/lox tissue-specificorinducibleknockouts.Togeneratemicethatareheterozygousfora loxP-flankedalleleandhemizygous/heterozygousforthe cre transgene,mateahomozygous loxP-flankedmouseofinteresttoacretransgenicmousestrain(seeFigure1below).Approximately50%oftheoffspringwillbeheterozygousforthe loxP alleleandhemizygous/heterozygousforthe cre transgene. Figure1 Matethesemicebacktothehomozygous loxP-flankedmice(seeFigure2below).Approximately25%oftheprogenyfromthismatingwillbehomozygousforthe loxP-flankedalleleandhemizygous/heterozygousforthe cre transgene.Thesewillbeyourexperimentalmice. Figure2 About25%oftheoffspringfromthismatingwillbehomozygousforthe loxP flankedallelebutwillhavenocre transgene.Dependingonyourexperimentalparameters,youcanusetheseascontrols(seeFigure3Abelow).BecausethecontrolswillnothaveCrerecombinase,anyphenotypicdifferencesbetweenthemandtheCre/loxmiceshouldbeduetothedeletedgeneintheCre/loxmice.Ifappropriateforyourexperiments,youcanmaintainacolonythatishomozygousforthe loxP flankedallele,butinwhichoneparentwashemizygous/heterozygousfor cre andtheotherwasanoncarrier/wild-typefor cre. ThisbreedingschememaynotbethemostefficientoneforproducingallCre/lox strains.Youwillhavetoadaptittothegenotypesyouwanttoproduceandthegeneticbackgroundsandcharacteristicsofthe loxP-flankedand cre strainsyouuse.ToverifythatCrerecombinaseexpressiondoesnotcontributetoaphenotypeofinterest,Isuggestusingthe cre strainitself(withoutany loxP-flankedalleles)asacontrol(Figure3B).Oneofmycolleagues,Andy,haswrittenanawesomeblogpostonhow Crerecombinase canproduceaphenotypeonitsown.Ihighlyrecommendthatyoucheckouthisblog. Figure3 TheuseoftheCre/loxPsystemforgeneratingtissuespecificorinducibleknockoutsisapowerfultoolformousegenetics,butitisonlyonepieceofthetotalpicture.Below,Iwouldtofocusontwoadditionalissues:usingCre/loxmicetoturnonorofftransgenes,andusingCrereporterstrains. ACreswitchfortransgenes TheCre/loxsystemisoneofthemostpowerfulandversatiletoolsdevelopedformousegenetics.Itgivesmouseresearcherssophisticatedcontroloverthelocationandtimingofgeneexpression.Cre/loxisusuallyusedtomakeknockoutalleles,butitcanalsobeusedtoactivategeneexpression. TheproperinsertionofaloxP-flanked“stop”sequence(transcriptionalterminationelement)betweenthepromoterandtransgenecodingsequenceblockstheexpressionofthegene.Forafewexamplesofsuchstrains,checkout008179and006481.Crerecombinaseremovesthestopsequence,sothetransgeneisexpressedonlyinthecellswhereCreisactive(seeFigure4below). Figure4 TheCre/loxsystemcanalsobeusedtoproducestrainsinwhichatransgeneiseitherinducibleorexpressedonlyincertaintissues.Forexample,matingthetransgenicstraininFigure1toastrainthatexpressesCrerecombinaseinmammarytissueproducesdoubletransgenicoffspringthatexpresstheKrasoncogeneonlyinthemammaryglands(Figure5).Noticethatonlyonegenerationofbreedingisrequired(Hooray!). Figure5 TheCre/loxsystemcanbeusedtoturnoffatransgene.SuchatransgeneisdesignedwithloxPsitesflankingallorpartofit.Asanexample,see008917.Inthisexample,expressionofCrerecombinaseremovesthetransgene(Figure6). Figure6 IfyouarelookingfortherightCre-expressingstrainforyourexperiments,youcansearchforstrainsattheJAXCreRepositoryorthroughtheCrePortalatMGI. Extra!Extra!ReadallaboutCreexpression! WhenresearchersstartedcreatingCrerecombinasemousestrainstogeneratetissuespecificknockoutmice,theyneededaneasywaytoconfirmthatCrerecombinasewasactiveonlyincertaintissues.ThisneedledtothedevelopmentofCrereporterstrains.Thesestrainsaredesignedtoexpressavisiblemarker,suchasgreenfluorescentprotein(GFP)orLacZ,onlyafterCrerecombinaseexcisesaloxP-flankedstopsequence(Figure7and8.) Figure7 WhensuchareporterstrainismatedtoaCre-expressingstrain,itproducesoffspringinwhichthevisiblemarkerisexpressedonlyintissueswithCreactivity(Figure8).Again,onlyonegenerationofbreedingisrequired. Figure8 Crereporterstrainscanalsobeusedtogenerateasourceoflabeledtissues/organs,whichallowresearcherstotrackcellsintransplantoradoptivetransferexperiments. MoreLikeThis Theprogressofgeneticsequencing Wetakealookatthehistoryofgenomicsequencing,whereit'sgoinginthefuture,andthepowerithastopotentiallytreatdiseaseslikecancer. ReadMore Navigatingprecisionmedicineincancer Withgenomictumortesting,anegativeresultisnotalwaysdefinitive.Insomecasesagenomicvariantcanbepresentbutnotdetectedbythe… ReadMore Assessingpatienthealthriskswithlimitedorunknownfamilyhistory WithNovemberdesignatedasFamilyHealthHistorymonth,cliniciansaresuretohaveseenlotsofmessaging… ReadMore Usingthepowerofsequencingonoursmallestorganisms Themicrobiomeplaysavitalroleinourhealthandwellbeing.Nowresearchersareusingsequencingtechnologytobetter… ReadMore