The gene targetingMario Capecchi.docx
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The gene targetingMario Capecchi.docx
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ThegenetargetingMarioCapecchi
Thegreatexperiments
Genetargeting:
Alteringthegenomeinmice
MarioCapecchi
MarioCapecchi
Genetargetingprovidesthemeanstocreatestrainsofmicewithspecificmutationsinvirtuallyanygene.Thismethodologypermitsevaluationofthefunctionsofgenesinanintactmammalandsystematicgeneticdissectionofthemostcomplexbiologicalprocesses,suchasdevelopment,learning,andimmunity.Theprimaryproblemindevelopinggenetargetingwashowtogenerateaspecificmutationinamammaliancellwithoutalteringanyothersiteinthegenome.Thisbecamepossiblewhenwediscoveredthatculturedmammaliancellsarecapableofhomologousrecombination,allowingamutantgeneinjectedintoacelltoreplacethechromosomalcopy.Paralleladvancesintheisolationofembryonicstemcellsmadeitpossibletoapplythistechniquetocellswithsufficientdevelopmentalpotentialtoallowmutationstobeintroducedintothemousegermlineandtheireffectsultimatelyobservedinwholeanimals.
Background
Ourcontributionstogenetargetingproceededinthreephases,thefirstbeginningin1977.AtthattimeIwasattemptingtoimprovetheefficiencywithwhichnewgenescouldbeintroducedintomammaliancells.WiglerandAxelhadjustreportedthefirstsuccessfultransformationofmammaliancellswithanexogenousgene,havingsucceededintransferringaherpessimplexvirusthymidinekinasegene(HSV-tk)intotk–culturedcellsbycalciumphosphatecoprecipitation(1985).Althoughanimportantcontributiontosomaticcellgenetics,thisprocedurewasnotefficient.Approximatelyoneinonemillioncellsexposedtothecalciumphosphate-DNAcoprecipitateacquiredtheexogenousgeneinafunctionalform.Usingthesameexperimentalparadigm,IaskedwhetherIcouldintroducetkgenesintotk–mousefibroblastsbyinjectingDNAdirectlyintotheirnuclei(1986).Thisprocedureprovedextremelyefficient.OnethirdofthecellsthatreceivedtheDNAstablyintegratedthefunctionaltkgeneatrandompositionswithintheirgenomesandpasseditontotheirdaughtercells.ThishighefficiencyofDNAtransferbymicroinjectionmadeitpracticalforinvestigatorstogeneratetransgenicmicecontainingrandominsertionsofexogenousDNAintheirgenomes.ThiswasaccomplishedbyinjectingthedesiredDNAintonucleiofone-cellzygotesandallowingtheseembryostocometoterminfostermothers(seeTransgenicmice:
Expressionofforeigngenesinanimals).
EfficientfunctionaltransferofthetkgeneintocellsrequiredthatitbelinkedtoothershortviralDNAsequences.Whenconsideringhowtoimprovetheefficiencyoftransformationofmammaliancells,IhadthoughtitplausiblethatviralgenomesmightcontainbitsofDNAthatenhancedtheirabilitytoestablishthemselveswithinamammaliangenome.IsucceededinfindingsuchasequenceinthegenomeofSV40,asimianDNAvirus.WhencoupledtotheHSV-tkgene,theSV40sequenceincreasedtheefficiencyofconferringatk+phenotypetotk–recipientcellsbyafactorofmorethan100(1986).TheenhancementdidnotappeartobetheresultofHSV-tkplasmidreplicationwithinthehostcellbeforeintegration,andtheSV40DNAsequenceswereintegratedintothehostgenomealongwiththeHSV-tkgene.Iconcludedthattheefficiency-enhancingsequencefromSV40waseitherincreasingthefrequencywithwhichtheexogenousDNAwasintegratedintothehostgenome,orincreasingtheprobabilitythatthetkgene,onceintegrated,wasbeingexpressed.TheSV40sequenceprovedtobewhatwouldlatercometobecalledanenhancer,andtheseexperimentscontributedtotheirdefinition(1987).Ourearlyexperiencewiththemwouldproveinvaluableindevelopingareliabletargetingapproach.
Figure 1
Aplasmidinjectedintoacellbecomesintegratedintoitsgenomeasaconcatemer.Twomechanismsarepossibleforconcatemerformation.
Althoughtheabilitytotransformcellsefficientlywasitselfextremelyuseful,theobservationthatIfoundmostfascinatingfromtheseearlyDNAmicroinjectionexperimentswasthatwhenmultiplecopiesofthetkplasmidwereinjectedintocells,theywerealwaysintegratedintothegenomeasheadtotailconcatemers.Thisstructureresultedeventhoughintegrationcouldapparentlytakeplacethroughoutthehostchromosomes.Irealizedthatconcatemerscouldbegeneratedeitherbyreplication(e.g.,arollingcirclemechanism)orbyhomologousrecombinationamongtheinjectedplasmids,asshowninFigure1.Wewereabletoprovethattheywereformedbyhomologousrecombination(1988).
Thiswasastartlingdiscoveryatthetime,becauseithadalwaysbeenassumedthathomologousrecombinationwasrestrictedtogermcells,whereitspurposewastoshuffletheparentalgenetictraitstoensuretheirbroaddisseminationamongtheoffspring.Findingevidenceofhomologousrecombinationinmousefibroblastsimpliedthatsomaticcellsalsopossessedthenecessarymachinery.Wesuspectedthatthehomologousrecombinationmachineryinsomaticcellswasveryefficient,becausewhenweinjectedmorethanonehundredtkplasmidmoleculespercelltheywereallincorporatedintoasingle,orderedhead-to-tailconcatemer.ItwasimmediatelyclearthatifwecouldharnessthismachinerytocarryouthomologousrecombinationbetweenanewlyintroducedDNAmoleculeofourchoiceandthesameDNAsequenceinarecipientcell'schromosome,wewouldhavetheabilitytospecificallymutateormodifyvirtuallyanycellulargene.
Thesecondphaseofourquestforgenetargetingrequiredthatwebecomefamiliarwiththesubstratepreferencesandreactionproductsofthecellularrecombinationmachinery.WedidthisbystudyingrecombinationbetweencointroducedDNAsubstrates.Theseexperimentsshowedthattheendogenousrecombinationmachinerycouldmediateawidespectrumofreactions.Althoughadistinctbiastowardsnonreciprocalreactionswasobserved,bothreciprocalexchangesandnonreciprocalrecombinationeventswereapparentamongtheproductsofrecombination(1989)(foradiscussionofrecombinationseeGenes2000BreakageandreunioninvolvesheteroduplexDNA.Eithertypeofreactionwouldsuitourpurpose.Wealsofoundthattheabilitytocarryouthomologousrecombinationdependsonacell'spositioninthecellcycle,showingapeakofactivityinearlyS-phase(1990),andthatlinearDNAmoleculesappearedtobebettersubstratesforhomologousrecombinationthancircularorsupercoiledmolecules(1991).Together,thesestudiesdemonstratedthatthecellularenzymaticmachineryforrecombinationnotonlyexisted,butthatwhenoptimizeditwassufficientlyefficientthatitmightindeedbeexploitedtomediatehomologousrecombinationbetweenexogenousDNAsequencesandchromosomalsequences.
Figure 2
Regenerationofafunctionalneorgenebygenetargeting.Thetargetingvectorandthechromosomecontaininactivatingmutationsatdifferentlocationswithintheneorgene.Homologousrecombinationbetweenthetwomutantgenesgeneratesafunctionalneorgenewithinthechromosomeinabout1in1000cells.
Thethirdphaseofoureffortwastodeviseawaytoextendhomologousrecombinationtositesinthegenomeitself.Tomakethingsassimpleaspossibleintheinitialstages,webeganwithstudiesinvolvinghomologousrecombinationwithspecifictargetsitesthatwehadcreatedwithinthechromosomes.Itwasclearpriortotheinitiationoftheseexperimentsthatthefrequencyoftargetingtoasinglecopygeneinmammaliancellswaslikelytobelowandthatinsertionofthetargetingvectoratsitesotherthanthetargetlocuswouldbefarmorecommon(simplybecausethereweresomanymoreofthem).Topermitdetectionofrarehomologousrecombinationevents,wedesignedlinesofrecipientcellswithspecifictargetlocithatwouldprovideaselectionprotocoltoeliminatecellsnotcontainingthedesiredrecombinationproducts,asoutlinedinFigure2.Thefirststepofthisschemerequiredgenerationofcelllinescontainingrandominsertionsofadefectiveneomycinresistance(neor)genecontainingeitheradeletionorapointmutation.Thesecondstepinvolvedintroducingintothosecelllinesatargetingvectorcarryinganeorgenewithaninactivatingmutationdifferentfromthatofthetargetneorgene.Homologousrecombinationbetweenthetargetingvectorandthecognatesequenceintherecipientcellgenomewouldgenerateafunctionalneorgenefromthetwodefectiveparts.CellscontainingthisrecombinationproductwouldberesistanttothedrugG418,whichkillscellsthatlackafunctionalneorgene.
Wegeneratedrecipientcelllinescontainingsinglecopiesofthedefectiveneorgene,linescontainingmultiplecopiesofthegeneinhead-to-tailconcatemersand,byinhibitingconcatemerformation,lineswithmultipledefectiveneortargetgenes,eachlocatedonaseparatechromosome.Thedifferentcelllinesallowedustoevaluatehowthenumberandlocationoftargetswithintherecipientcell'sgenomeinfluencedthetargetingfrequency.
Acquisitionofneomycinresistancebycellsinjectedwithaneortargetingvectoroccurredatafrequencyhigherthanweanticipated(1991).Oneinathousandinjectedcellsyieldedprogenywithfunctionalneorgenes.Southerntransferandsequenceanalysisshowedthattheneomycin-resistantphenotypewasindeedduetocorrectionofthedefectivechromosomalneorgenebyhomologousrecombinationwiththeincomingDNA,aswehadintended.Correctionofapointmutationbygenetargetingoccurredatafrequencythatwasfiveordersofmagnitudehigherthanthespontaneousreversionfrequency.Correctionofadeletionmutationwaseffectivelyataninfinitelyhigherfrequencyrelative
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