Flox Genes and Mice - How to Obtain a Conditional Knockout

The Cre-lox system allows for the deletion of a gene in mouse ES cells and its inversion or complete inactivation. This process is called “floxing” since it ... Flox January28,2019|ingenious|Cre-lox HowtoObtainaConditionalKnockout–FloxedMiceandtheCre-LoxProcedure Conditionalknockoutshavebecomeveryimportantresearchmodelswithinthelasttwodecades.Havinghelpedresearchersdiscoverthereasonsbehindcountlessailmentsandsomeoftherarestandmostdangerousgeneticdiseasesinhumans,geneticresearchhascomealongwayinthepast20yearsalone.WiththeCre-loxsystem,developedandperfectedoverthecourseofthe1990sand2000s,floxedmicehavebecomeamajortoolforgeneticresearchers.Theyusethesemicetostudy,documentandunderstanddiseasesandthepossiblecuresandtreatmentstheymightdevelopinordertocounterthem. Beforeunderstandingwhyfloxmicearesoimportant,itisnecessarytotakeacloserlookatthecomplexprocessthatmakestheirexistencepossible:theCre-loxrecombinationsystem.Thissystemisresponsiblefordevelopingfloxmiceinafractionofthetimethatwaspreviouslyusedtodevelopknockoutmice,duetoitssimplicityandimprovedefficiency.AlthoughtheCre-loxsystemisn’tperfect,Cre-loxtechnologycomesveryclose,asitcanproducesomeoutstandingresultsintherighthands.Thatbeingsaid,therearemanymorewaystocreatefloxgenesthantheusual,conventionalmethodsusedbybeginningresearchers. Cre-LoxTechnologyandtheDevelopmentofFloxGenesandMice TheCre-loxsystemallowsforthedeletionofageneinmouseEScellsanditsinversionorcompleteinactivation.Thisprocessiscalled“floxing”sinceitcombinesflankingthegenewiththehelpofitsadjacenttwoloxPsitesandtheCre-loxrecombinationsystem.WhileCrecanbeusedtostudyahugevarietyofgenefunctions,aswellasvarioustreatmentsanddiseasesthataresimilarinbothmiceandhumans,thatisnottheextentofitslimits.TheCresystemcanalsobeused,forexample,torecombinegenes,aswellastoconvertfloxedallelestoknockouts.It’spossibletocombinetheCresystemwithothergenemanipulationtechniquessuchastheTetsystem.Thelatterprovidesremarkablecontroloverthelocationandtimingofacertaingeneexpression.Furthermore,itcanbeusedtogetherwithfloxgenestoimprovetheprocessofspecifyingthetissueandtimewherethegenewouldoperate. Mostnotably,thegenerationoffloxmiceusingCre-loxtechnologycanleadtothedevelopmentofnewtypesoftissue-specificstrainsofmice.Althoughnotalwayspreciseorfunctionalforpracticalpurposes,thisbranchofgeneticresearchcanstillhelpscientistsmakenewdiscoveriesingeneticmanipulation.MatingCremicewithcertaintransgenicmicecanleadtoachangeinthecolorofthetargetedtissueoreventheexpressionofbeta-galactosidase(LacZ),luciferaseand/orphosphatase.Theseapproachesmightbemoreofacuriosity,buttheycanhelpagreatdealwhenitcomestogainingadeeperunderstandingofhowcertaingenesworktoinfluenceorchangetissue-relatedproperties,aswellashowtheymighthaveanimpactonthebodyasawhole. Generatingconditionalknockoutmice,however,isamuchmorecomplicatedmatter.AsidefromsimplyusingCre-loxtoremovethegenefromthesequenceandrenderingitunabletoperformitsfunction,thisknockoutprocessusingtheCre-loxmethodalsohastobeconditional–meaningthatithastobetriggeredatacertaintime.Thisimportantdetailmakesthesemiceevenmoredifficulttoobtain,astheprocessrequiresnotonlytheflankingofthegeneregionwiththehelpofCre-lox,butalsocrossingamousewiththatalleleinordertoobtainaspecificCre-expressingmouse.ItisthenpossibletouseinducibleCrewhichrecombinestoturnoffthegene. Therearemanyreasonsforscientiststousetheconditionalknockoutprocedure,alsoknownasgenedisruption,regularlyingeneticresearch.Itispossibletouseittoanalyzefatalgenesordisruptorgan-specificgenesinordertostudythelongandshorttermeffectsonthebody.Otherpossibilitiesincludeusingnewlydevelopeddrugsandmedicationstoattempttotreatadiseasethatoriginatesfromorresultsintheimpairmentofacertaingene.Withthehelpoffloxedmice,othergenesthatarespecifictocertainstagesofdevelopmentinthebodycanalsobestudied.Inaddition,treatmentmethodsandtheirlongtermeffectscaneasilybemonitoredinashortertime. ThebasicprocessofgeneratingafloxknockoutmousemodelusingtheCre-loxmethodseemssimpleenough,butitcanbequitechallenging.First,it’scrucialtounderstandhowtochoosetheproperCrestraininordertogenerateknockoutmice.Next,theregulationofthetimingandlocationofthegenerecombinationisestablished.ThetargetgeneisflankedattwoloxPsites,thentheCreexcisionismade,effectivelyisolatingandseparatingthegene.Then,asite-specificenzymeisusedtocatalyzetherecombinationprocessbetweenthetwoloxPsites.TheCre-loxdeletionprocessisthemostimportanttoolhere,asitallowstheproceduretoremainrathersimpleandfortheproductionofthefloxedmicetobemuchfasterandmoreaccurate. Ofcourse,Cre-loxtechnologyisnotperfect.Despiteitsmanybreakthroughsandtheabilityitprovidesresearcherstoeasilygeneratethefloxmicetheyneed,thestrategyisstillsomewhatflawedbecauseofthelackofknowledgeaboutallthepotentiallyusableinsertionsitespresentinsidetheDNA.Becauseofthis,itcanbeagamblewhenusinghemizygousmiceinordertocreatefloxmousemodelsofacertaingene,especiallysincehemizygousmicedon’thaveawildtypechromosomepresent,ashomozygousmicedo.Asaresultofthisfact,homozygousmicearebeingusedtoafargreaterextentandwithmoreaccurateresults.BecauseCre-loxcanimpairtheDNAinfibroblasts,spermcellsandgastriccells,newerresearchersareusuallyadvisedtoincludetheCremouseitselfasacontrolinanystudiestheywanttoconductusingfloxmice.Withthehelpoffloxedmice,geneticresearchershavefarmoreoptionswhenitcomestoaccuratelytargetingandeliminatingtheirgeneofinterest,allowingforawiderrangeofstudies. Sharethis: Facebook Twitter LinkedIn Utilizingingenioustogeneratemymouseknockoutvalidatedmydecisiontonottryanddothisinmylab“Theprojectwasverywellmanaged…infact,usingiTLvalidatedmydecisiontonottryanddothisinmyownlab.Itwouldhavebeenacatastrophe…(Myproject[…]ClausFimmel,MDLoyolaUniversityMedicalCenterReadmoreSimilarArticles