Transgenic Mouse Models in Cancer Research - Frontiers

Transgenic mice, in which the gene is depleted or silenced to cause a loss of gene function, are called knockout mice. These mice provide ... ThisarticleispartoftheResearchTopic CancerModels Viewall 14 Articles Articles MichaelBreitenbach UniversityofSalzburg,Austria ReinhardUllmann InstitutfürRadiobiologiederBundeswehr,UniversitätUlm,Germany MartinHolcmann MedicalUniversityofVienna,Austria Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction ProductionofTransgenicMice TypesofTransgenicMice NewMouseModelsforCancerResearch CurrentDirectionsinTransgenicMouseCancerModels Conclusion AuthorContributions ConflictofInterestStatement Acknowledgments References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData REVIEWarticle Front.Oncol.,20July2018 |https://doi.org/10.3389/fonc.2018.00268 TransgenicMouseModelsinCancerResearch UrsaLamprehtTratar1, SimonHorvat2andMajaCemazar1,3* 1DepartmentofExperimentalOncology,InstituteofOncologyLjubljana,Ljubljana,Slovenia 2BiotechnicalFaculty,UniversityofLjubljana,Ljubljana,Slovenia 3FacultyofHealthSciences,UniversityofPrimorska,Isola,Slovenia Theuseofexistingmousemodelsincancerresearchisofutmostimportanceastheyaimtoexplorethecasuallinkbetweencandidatecancergenesandcarcinogenesisaswellastoprovidemodelstodevelopandtestnewtherapies.However,fasterprogressintranslatingmousecancermodelresearchintotheclinichasbeenhamperedduetothelimitationsofthesemodelstobetterreflectthecomplexitiesofhumantumors.Traditionally,immunocompetentandimmunodeficientmicewithsyngeneicandxenograftedtumorstransplantedsubcutaneouslyororthotopicallyhavebeenused.Thesemodelsarestillbeingwidelyemployedformanydifferenttypesofstudies,inpartduetotheirwidespreadavailabilityandlowcost.Othertypesofmousemodelsusedincancerresearchcomprisetransgenicmiceinwhichoncogenescanbeconstitutivelyorconditionallyexpressedandtumor-suppressorgenessilencedusingconventionalmethods,suchasretroviralinfection,microinjectionofDNAconstructs,andtheso-called“gene-targetedtransgene”approach.Thesetraditionaltransgenicmodelshavebeenveryimportantinstudiesofcarcinogenesisandtumorpathogenesis,aswellasinstudiesevaluatingthedevelopmentofresistancetotherapy.Recently,theclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)-basedgenomeeditingapproachhasrevolutionizedthefieldofmousecancermodelsandhashadaprofoundandrapidimpactonthedevelopmentofmoreeffectivesystemstostudyhumancancers.TheCRISPR/Cas9-basedtransgenicmodelshavethecapacitytoengineerawidespectrumofmutationsfoundinhumancancersandprovidesolutionstoproblemsthatwerepreviouslyunsolvable.Recently,humanizedmousexenograftmodelsthatacceptpatient-derivedxenograftsandCD34+cellsweredevelopedtobettermimictumorheterogeneity,thetumormicroenvironment,andcross-talkbetweenthetumorandstromal/immunecells.Thesefeaturesmakethemextremelyvaluablemodelsfortheevaluationofinvestigationalcancertherapies,specificallynewimmunotherapies.Takentogether,improvementsinboththeCRISPR/Cas9systemproducingmorevalidmousemodelsandinthehumanizedmousexenograftmodelsresemblingcomplexinteractionsbetweenthetumoranditsenvironmentmightrepresentoneofthesuccessfulpathwaystopreciseindividualizedcancertherapy,leadingtoimprovedcancerpatientsurvivalandqualityoflife. Introduction Themouseasamodelforhumancancerresearchhasproventobeausefultoolduetotherelativelysimilargenomicandphysiologicalcharacteristicsoftumorbiologybetweenmiceandhumans.Micehaveseveralsimilaranatomical,cellular,andmolecularcharacteristicstohumansthatareknowntohavecriticalpropertiesandfunctionsincancer.Additionally,theproportionofmousegeneswithahumanorthologis80%(1),thusprovidinganexcellentexperimentallytractablemodelsystemasaresearchtooltoinvestigatebasicmechanismsofcancerdevelopmentaswellasresponsestotreatment(2).Althoughconventionaltransgenicmousemodelshaveremainedavaluabletooltoexaminethemolecularmechanismsofcarcinogenesis,alimitationhasbeenalowdegreeofheterogeneityinmousetumorsincomparisontotheveryheterogeneoushumantumors.Severaladvanceshavebeenmadeinmodelingcancerinmice,andthenewmodelsdescribedinthisreviewarenowmorecapableofmodelinghumancancerswithmutationsthatarecontrolledspatiallyand/ortemporally.Inaddition,thesemodelsbetteraddresstumorheterogeneityandinter-patientvariabilityintheclinicalsetting(3). Traditionally,immunocompetentandimmunodeficientmicewithsyngeneicandxenograftedtumorstransplantedsubcutaneouslyororthotopicallyhavebeenused.Thesemodelsarestillwidelyappliedformanydifferenttypesofstudiesandarealsoaffordable(4).However,intheearly1980s,newtypesofmousemodelsemergedwithengineeredmutationsintheirgenomethatrevolutionizedcancerresearch.In1974,R.JaenischandB.Mintzperformedanexperimentwhereintheviraloncogenesfromsimianvirus(SV40)weremicroinjectedintotheblastocoelofmouseembryos.Althoughtheresultingmicedidnotdeveloptumors,theycoulddetecttheviralDNAintegratedinthegenomeofcellsofmanydifferenttissues.Thesemiceareconsideredthefirsttransgenicmice(5).Laterinthe1980s,thefirsttransgenicmousecancermodelswereproduced,whichweregeneticallyengineeredtoexpressdominantoncogenes.Withtheseso-called“oncomice,”thepredispositionsrequiredforthedevelopmentofcancer,aswellasnewtargetsforthedevelopmentofnoveltherapeuticapproaches,couldbeinvestigated.Later,moredefinitivemodificationsinthegenomewereperformedwithknockoutandknock-inmice,andsincethenseveralresearchpapershaveusedthetermtransgenicmiceasadistinctgroupfromknockoutandknock-inmice(6).Duetotheconfusionrelatedtothenomenclature,in2007,theFederationofEuropeanLaboratoryAnimalScienceAssociationsreleasedguidelinesfortheproductionandnomenclatureoftransgenicrodents.Intheseguidelines,itisstatedthattransgenicanimalsarereferredtoasthosewithspontaneous,chemicallyinducedmutationsandthosewithrandomorgene-targetingDNArecombinationevents(7).TheNationalInstituteofHealth,NationalCancerInstitute(NIHNCI)referstothetermtransgenicanimalsformodelsinwhichDNAfromthemousegenomeorfromthegenomeofanotherspecieshasbeenincorporatedintoeachcellofthemousemodelgenome(8).Thesemicearenowmostlycalledtransgenicmice,buttheyarealsoreferredtoasgermlinegeneticallyengineeredmousemodels(GEMM),includingknock-inandknockoutmousemodels(9).Additionally,themousegenomeinformaticsdatabase(10),themaindatabaseresourceforthelaboratorymouse,listsmutantallelesforeachgeneinseveralcategories,including“transgenicmodels”similartothedefinitioninreference8aboveand“targeted,”whichencompassesbothtargetedknockoutandknock-inmodelssimilartothedefinitionofGEMMabove.Inthisarticle,werefertotheterm“transgenic”asageneraltermforalltypesofgenomealterationsinthegermorsomaticcellsand/orinvitroandinvivomousemodels. ProductionofTransgenicMice TransgenicmicecanbeproducedinseveralwaysbyintroducingDNAintothemousegenome(Figure1). 1.Byretroviralinfectionofmouseembryosatdifferentdevelopmentalstages.Thismethodisnotroutinelyusedfortheproductionoftransgenicmice(11),inpartduetothesilencingofthetransgeneofviraloriginfollowingdenovoDNAmethylationafterinsertionoftheretroviralvector(12).Anotherlimitationisalsoarelativelysmallsizeoftheinsertthatcanbecarriedbythevector,aswellasrandomintegration,whichcaninfluencetheexpressionoftheneighboringgenes,resultinginphenotypesthatareunrelatedtothetransgene. 2.BymicroinjectionofDNAconstructsorrecentlybymicroinjectingendonuclease-basedreagents(e.g.,Cas9–sgRNA–ssDNAmixture)directlyintothepronucleusoffertilizedmouseoocytes.First,inthecaseofinjectingDNAconstructs,thetransgeneisrandomlyintegratedinasmallpercentageofinjectedoocytesasoneormoretandemcopiesintothemousegenome,andgenerallyallthecellsofsuchoffspringpossessthetransgene(13–15).Themethodtoproducetransgenicprogenyisrelativelyquick,butitincludestheriskthattheDNAmayinsertintoacriticallocusthatcancauseanunexpected,detrimentalgeneticmutation.Second,thetransgenemayinsertintoalocusthatissubjectedtogenesilencing(16).Third,iftheDNAconstructinsertsasmultipletandemcopies,itcanproduceextremeoverexpressionleadingtonon-physiologicalphenotypiceffects,butmoreoftensuchtandemtransgeneintegrationsaresilencedinsubsequentgenerations.Inthecaseofusinganewendonucleaseapproach,reagentsarealsomicroinjectedintothefertilizedeggs,butherethegeneticmodificationisproducedatatargetedsitealbeitalsowithsomeoff-targetevents.Moreonthisnovel,CRISPR-Cas9-basedmethodisdescribedbelow. 3.Thethirdapproachiscalledthe“gene-targetedtransgeneapproach.”Itincludesthetargetedmanipulationofmouseembryonicstem(ES)cellsatselectedlocibyintroducingprimarilyloss-of-functionmutations(11).GeneticallymodifiedEScellsarethenmicroinjectedintothemouseblastocystsandtransferredtopseudopregnantrecipientmice.TheEScellsanddonorblastocystsderivefrommouselineswithdifferentcoatcolors,andthussuccessfulincorporationoftargetedEScellsintothedevelopingembryoofdonorblastocystresultsinchimericoffspringexhibitingvariegatedcoatcolor.Chimericoffspringarefurthermatedwithwild-typemicetotestforgermlinetransmissionofthetransgene.Ifchimericmiceshowingvariegatedcoatcolor(meaningtheyaresomaticchimeras)arealsogermlinechimeras,thenthecrosswithwild-typemicewillresultinacertainpercentofheterozygousprogenycarryingthetransgene.Finally,theintercrossofsuchheterozygotesproduceshomozygousmutantmiceatanexpected25%frequencyunlessthemutationisdetrimentaltoembryosurvivalanddevelopment(11,17,18).Variousexperimentsarethencarriedoutonmutanthomozygotestotestthefunctionalityofthegeneticmodification. FIGURE1 Figure1.Differentstrategiesforcreatingtransgenicmiceincludethe(A)retroviralapproach,whichisnotroutinelyused;(B)standardtransgeneapproach,inwhichtheDNAisinsertedintothegenomeinanunspecificmanner;and(C)gene-targetedtransgeneapproach,whichisanapproachthatisroutinelyusedtocreateconventionalknockouttransgenicmice,usuallywithaconstitutiveloss-of-functionmutation. TypesofTransgenicMice Theproductionoftransgenicmicedescribedaboveistime-consumingasitcantakeseveralyearstoestablishamutationintheEScellsanddevelopandvalidateanewtransgenicmousemodel.Nevertheless,traditionaltransgenicmicearewidelyusedinpreclinicalresearchinoncologyaswellasinotherresearchfields.Oneofthemaindrawbacksofusingtransgenicmiceinpreclinicalresearchisthelongtimerequiredtogeneratenewtransgenicmouselines.Forexample,theproductionoftransgenicmicebygenetargeting(Figure1C)requiresveryefficienttargetingofEScells,thegenerationofgermlinechimeras,atleasttwogenerationsofcrossestoobtainhomozygotes,colonyexpansionofhomozygousgene-targetedmiceandonlythencanthecharacterizationofoncologicalphenotypesbeperformed.Ifthegene-targetedmutationisdominant(i.e.,heterozygotesexpresstheoncologicalphenotype),theprocedureisonegenerationshorterbutstilllong.InallthemethodsdescribedinFigure1,theprocessofgeneratingandcharacterizingtransgenicmicetakesseveralyears,andinadditiontobeingtime-andlabor-consuming,italsorequiressubstantialfinancialsupport.Inaddition,oneoftheshortcomingoftraditionaltransgenicmousemodels(Figures1A,B)isthatasubstantialfractionofmicecanexhibitheterogeneityintheirphenotypes,includingdifferentialtissueorcelltypeexpressionorthegenerationofadditionalphenotypesthatarenotrelatedtothetransgeneduetothesiteinthegenome-integrationeffects,wherebythetransgeneaffectstheexpressionofneighboringgenesorepigeneticallyaltersalargerregionincis.Thisadditionalvariabilityinphenotypesresultsinanincreasednumberofmicethatarerequiredtogeneratethetransgenicmousemodelthatisstable(19).However,thisphenomenonisnotinlinewiththe3Rprinciples,especiallytheprinciples“reduce”and“refine”(20).Forallthesereasons,newwaysofgeneratingtransgenicmousemodelshaveemerged,notonlywithalternativewaysofmodifyingDNAthatwillbediscussedlaterbutalsowiththeuseofnon-germlinegeneticallyengineeredmousemodels(nGEMM)thatdonotcarryDNAmodificationsingermlinecellsbutonlyinsomesomaticcells.Inaddition,classicaltransgenicmousemodelscanbeimprovedbyinducibleorconditionaltransgenesistechniques. Roughly,transgenicmicecanbedividedintotwogroupsconsideringthelossorgainoffunction. LossofFunction Transgenicmice,inwhichthegeneisdepletedorsilencedtocausealossofgenefunction,arecalledknockoutmice.Thesemiceprovidevaluablecluesaboutthebiologicalfunctionofanormalgene.Intranslationalcancerresearch,thisrepresentsapowerfultoolinassessingthepotentialvalidityoftargetedtherapybecausethetargetscanbepreciselyinactivatedinthesettingofadevelopingordevelopedtumor.Inaddition,studiesusingknockoutmiceareimportanttoelucidatethecauseandeffectrelationshipincancerdevelopment.Suchstudieswithknockoutmicecanbeappliedfortheassessmentofmanygeneclasses,includingoncogenes,tumor-suppressorgenes,andmetabolic(“housekeeping”)genes(3).Theloss-of-functionmodelscanalsoincludedominant-negativetransgenicmousemodelsthatcarryspecificmutationsthatdisrupttheactivityofthewild-typegeneeitherbyoverexpressionorothertypesofmodificationsofgenestructure.Forexample,dominant-negativetransgenicmicehavebeenusedtoprobethefunctionofE-cadherinanditscausalroleinthetransitionfromadenomatocarcinoma(21). Twotypesofknockoutmousemodelsarefrequentlyused:constitutive(permanentlyinactivatedtargetgeneexpressionineverycelloftheorganism)andconditional(inducibleinactivationofgeneexpression),whichcanaffectaspecifictargettissue(tissueorcelltypespecific)orcanoccurinatime-controlledmanner(temporal)(22). ConstitutiveKnockout AconstitutiveknockoutmousegeneratedbyaproceduredescribedinFigure1Cisoftenreferredtoasaconventionalorwhole-bodyknockoutmodel.Itdefinesamousemodelinwhichthetargetgeneispermanentlyinactivatedinthewholeanimal,ineverycelloftheorganism(23).Thesemousemodelscanbeusedtoassessthechangesinamouse’sphenotype,suchasanatomy,physiology,behavior,andotherobservablecharacteristics.Incancerresearch,knockoutmousemodelshavebeeninvaluablefortheidentificationandvalidationofnovelcancergenes.Forexample,constitutiveknockoutmodelswereusedtoidentifytheroleofthenewlydiscoveredgenesushidomaincontaining6[Susd6,humansynonym;drug-activatedgeneoverexpressed(DRAGO)]asanewp53-responsivegeneinducedafterthetreatmentwithdrugsthatinterferewithDNA.TheresultsofthatstudyshowedthatdeletionofbothDragoallelesinp53−/−orp53+/−micecausedstatisticallysignificantlyacceleratedtumordevelopmentandashortenedlifespancomparedwiththatofp53−/−orp53+/−micethatborewild-typeDragoalleles(24).Nevertheless,theuseofconstitutiveknockoutsincancerresearchislimitedbecausetheydonotimitatethesporadicdevelopmentofatumorgrowingfromasinglecellinanotherwisenormalenvironment(clonalevolutionoftumors).Additionally,simpleknockoutsarefrequentlyintendedtoleadtoalossofproteinfunction(andlatelyinnon-codingRNAgenes),whereasincancer,asubsetofcancer-causingmutationsconsistentlyalsoresultsinagainoffunction(25).Furthermore,oneofthemajordrawbacksofconstitutiveknockoutsisthatgermlinelossoffunctionoftenleadstoembryoniclethality,severedevelopmentalabnormalitiesoradultsterility,makingconclusivedeterminationsabouttumor-suppressorgenesmoredifficult(26). ConditionalKnockout Duetothelimitationsoftheconstitutiveknockoutmodel,modificationoftheknockoutmodelemergedtoleadthedevelopmentofconditionalknockoutmodels.Theconditionalknockoutmodelcanmoreefficientlymimicspontaneouscarcinogenesisbecauseinhumans,tumorsevolveinawild-typeenvironment,andtherefore,thetimingofgenelossmaybeacriticalfactorindiseasedevelopment(3).Thus,toavoidand/orimprovethelimitationsoftheconstitutiveknockout,conditionalmodels,inwhichthegeneknockoutcanbespatiallyandtemporallyregulated,weredeveloped.ThemainactorsinthistechnologyarebacterialCreandyeastFLPenzymes,whichactassite-specificrecombinasestocatalyzerecombinationbetweenspecific34-bploxPandFRTsites,respectively.WhenCreorFLPproteinsareexpressed,homologousrecombinationisinducedbetweenloxPorFRTsites.Thesesitesflankthegeneofinterestandareorientedinthesamedirection,whichcausesthedeletionofthegeneofinterestafterrecombinationofflanksofgeneticsequence.Expressionoftherecombinasecanbecontrolledtemporallyorspatially,andtherefore,wecancontrolthedeletionofthegeneofinterestintemporalandspatialmanners,thusovercominginterferencesduetodevelopmentalabnormalitiesandlethality(27). Forspatialcontrol,micecarryingtheCreorFLPrecombinaseunderthecontrolofatissue-specificorinduciblepromotormustfirstbedeveloped,usuallyviathemethoddescribedinFigure1B.Whenthesemicearecrossedwithgene-targetedmicecarryingthegeneofinterestflankedbyloxPorFRTsites(developedviatheprocedureinFigure1C),thetargetgeneintheprogenycanbeconditionallyinactivatedinaspecifictissueorcelltypeoratspecifictimesduringdevelopment(3).Tissue-specificknockoutmodelscanalsobeproducedbyviraldriveninduciblevectorsdeliveredlocallyortopicallybyinjectiontoinfectthecells,therebydeliveringCreorFLPenzymestotargettissuesorcells.Thismethodcreatesregionalknockoutofcellswithintheappliedarea(28).Bothadenovirusandlentivirusvectorscanbeused(29). ForthetemporalcontrolofCreexpression,tetracyclineandtamoxifen-induciblesystemsaremainlyused(30).Inthecaseoftetracycline-basedsystem,atransactivatorandaneffectorareused.Thetetracycline-controlledtransactivator(tTA)proteinbindstothetetracyclineoperator(tetO)thatcontrolstheactivityofCreexpressiontogenerateconditionalknockouts.Whenaddingtetracyclinetotheanimal’sdrinkingwater,theingesteddrugbindstotTAandinhibitstheassociationwithtetO,blockinggenetranscription(31).ThisiscalledaTet-offsystem,whereingeneexpressionisinhibitedinthepresenceoftetracycline.WhenusingtheTet-onsystem,reversetetracycline-controlledtransactivator(rtTA)proteinbindstotetOonlyifitisboundtotetracycline.Therefore,thepresenceoftetracyclineintheanimalinitiatesgeneexpression(32).OneoftheshortcomingofthissystemistheleakinessofrtTA,whichcompromisesthedesiredregulationoftransgeneexpression.ThertTAmaintainssomeaffinityfortetOsequencesevenintheabsenceoftetracycline,whichresultsintheundesiredtranscriptionoftargetgenes(33). Inthetamoxifen-induciblesystem,theCrerecombinasegeneisfusedtoamutatedligand-bindingdomainofthehumanestrogenreceptor(Cre-ER(T))thatisspecificallyactivatedbytamoxifen.Whenactivetamoxifenmetabolite4-hydroxytamoxifenisabsent,theERfusionproteinisexcludedfromthenucleus.Afterbindingtotamoxifen,theERfusionproteinisagaintransportedtothenucleus,enablingthebindingofCrerecombinasetoDNA.Therefore,temporalexpressionofCrecanbecontrolledbydeliveringorwithholdingtamoxifentotheanimals(34).Conditionalknockoutmousemodelhavebeenusedinmanydifferentstudies,includingthosemanipulatinggenes,suchasK-Ras,Myc,andp53(25),aswellasinstudiesevaluatingtumor-initiatingcells.Forexample,thedevelopmentofabnormaldifferentiatedSchwanncells,whichserveasneurofibromatumor-initiatingtumorcells,hasbeenshowntoresultfromtheconditionallossofNf1infetalneuralcreststem/progenitorcellsoftheSchwanncelllineage(35).Onelimitationofthetamoxifen-induciblesystemistheleakinessoftheCre-ERmodels,whichcausesacertainlevelofnucleartranslocationofCre-ERevenintheabsenceoftamoxifen(36).Suchaneventcancauseanundesiredgainorlossoffunctionalmutations(37). Likeanystrategy,theproductionofconditionalknockoutmodelsalsohasdrawbacksandlimitations:theprocedureusedtodevelopthesemodelsislengthyandrequiresadditionaltransgenicCreorFLPtransgenicmodels,withthepossibilityofmosaicexpressionofCreorFLP-driventransgenesasmanyCrelinesarepronetobothtemporalandspatialectopicexpression,geneticbackgroundeffects,oreveneventualsilencingoftheexpressionofCreorFLP-driventransgenesinmiceinlatergenerations(38).However,comparedwiththeconstitutiveknockout,conditionalknockoutmutagenesisisadvantageousbecauseitusessubtlergeneticmodificationstoexaminethefunctionalrole(s)ofgene(s)inatissueortemporalmanner.Italsoavoidspotentialembryoniclethalityfromtheconstitutiveknockoutapproach,makingitpossibletostudyessentialgenes. GainofFunction Gain-of-functionstudiesareoftenusedtostudyoncogenesinmousemodels.Knock-inmodelsofoncogeneoverexpressioncanbeusedtostudyhowtheoncogenedrivescarcinogenesisinvivo. ConstitutiveRandomInsertionModel Theconventionalrandominsertionmousemodelcanbeproducedbyviralvector-basedtransfectionofearlymouseembryosorbypronuclearinjectionofthetransgenedirectlyintofertilizedoocytes(Figures1A,B).Thetransgeneisthenrandomlyincorporatedintothegenome.Althoughtheprocedureisverystraightforwardandrelativelysimple,therandomincorporationintothegenomeisthemaindrawbackofthismodelbecauseitcanresultinanundesirableexpressionlevelorspatiotemporaldistributionoftransgeneactivity,orevendeleteriouseffects,thuslimitingtheusefulnessofthemodel.ThesemodelshavebeenwidelyusedtostudyhowoncogenessuchasK-rasdrivetumorigenesisinvivo(39–42). Knock-inPermissiveLocusModel Toovercomethelimitationsoftheconstitutiverandominsertionmodel,severalnewmodelshavebeendevelopedtostudythegainoffunction,specificallybyinsertingageneofinterestintoaspecificregionofthegenome.Usinghomologousrecombination,amorepredictableandstablegain-of-functionmodelcanbeobtained.ThemostcommonlyusedsiteistheRosa26locusbecauseitdoesnotcontainanyessentialgenesandprovidesstableandpredictableexpressionofthetransgeneinvariouscelltypes(43,44).Npm1transgenicmicecanserveasagoodexampleofamousemodelusingtheRosa26locusandCre-regulatedexpression.TheNpm1mutation,whichisthemostfrequentgeneticalterationinacutemyeloidleukemia(AML)(45),canbecharacterizedinthisknock-inpermissivemodel.Withthismodel,ithasbeenshownthatNpm1mutationsaffectsmegakaryocyticdevelopmentandmimicssomefeaturesofhumanNPM1-mutatedAML,thusservingasagoodmodelforfurtherinvestigationsofAML(46). ConditionalKnock-InModel Aspreviouslypointedout,theconstitutivegeneknock-indescribedinSection“ConstitutiveRandomInsertionModel”canleadtolethality,sterility,anddevelopmentaldefects.Therefore,similartotheknockoutmousemodels,spatialandtemporalcontrolofthegenehastoberegulatedtoalsocircumventtheselimitationsinknock-inmodels.Conditionalknock-inmodelscanbegeneratedusingtissue-specificpromotorsorbyinsertingastrongtranslationalandtranscriptionaltermination(STOP)sequenceflankedbyloxPorFRTsitesbetweenthepromotorsequenceandthegeneofinterest(47).WhentheSTOPsequenceispresent,transcriptionofthegeneinterestisblocked.However,whenCreorFLPrecombinaseareexpressedandpresent,theSTOPcassetteisremoved,allowingexpressionofthegene(28).Thus,geneexpressionismediatedbyexcisionoftheSTOPcassetteandrecombinaseexpression.Therefore,geneexpressionisspatially,temporally,andinduciblymediatedbyCreorFLPsystems(48).Occasionally,alsodependingontheknock-ingenomesite,theSTOPcassettescanbeleaky,asobservedintheinitialK-rasG12Dmodelsoflungcarcinoma,whereindeathduetorespiratoryfailurepriortotumorprogressionoccurred(49).ImprovedSTOPcassetteswithlessleakinessweresubsequentlydeveloped.Laterversionsofconditionalknock-inmousemodelofLSL-K-rasG12Dwereshowntobegoodmodelstostudytheinitiationandearlystagepulmonaryadenocarcinoma,allowingcontrolofthetiming,location,andnumberoftumors(28).Additionally,conditionalknock-inmousemodelswerealsousedtoinvestigatetheroleofBrca1RINGfunctionintumorsuppressionandtherapeuticresponse,whereitwasdeterminedthatBrca1RINGdidnotaffectresistancetotherapy(50). ReporterKnock-InModel Toobservetheexpressionofthetargetedgeneatthetranscriptionalortranslationallevel,reporterknock-inmousemodelscanbeused.Genesencodingfluorescenceproteinshavebeenwidelyusedasreportersinbiomedicalresearchandfrequentlyemployedtoanalyzethetransgeneactivity.Inreportermodels,transgenesareusedforthevisualizationofproteomic,metabolic,cellular,orgeneticeventsinvivo.Themostcommonlyusedtechniqueforvisualizationisfluorescentandbioluminescentopticalimagingduetotheincreasedsensitivity,relativeinexpensiveness,andlesstime-consumingandmoreuser-friendlyfeaturescomparedwiththoseof,forexample,histological,genetic,orbiochemicalmethods.Furthermore,suchmodelsarealsoinlinewiththe3Rprinciplesinresearchusinganimals,incorporatingatleasttwooftheseprinciples;reduction(lessanimalsused)andrefinement(lessharmfulmethods)(20).Severaltransgenicmouselinesareavailablethatexpressreportergenes(51).Themostcommonreportersaregreenfluorescentprotein(GFP)andredfluorescentprotein(RFP)(52,53)forfluorescenceandfireflyluciferaseforbioluminescence(54).Thelattercanalsobeusedforthevisualizationoftumorgrowthinvivo.Tostudytumorcellproliferationinvivo,miceexpressingfireflyluciferaseundercontrolofthehumanE2F1genepromotor,whichisactiveduringproliferation,werecrossedwithamousecancermodel(55). Recently,incancerresearch,detectionandimagingoftheimmuneresponsehasbecomeoneofthefundamentalwaystofollowthetreatmentcourseinliveanimals.Becauseseveralnewtreatmentsaimtomodulatetheimmuneresponse,therecruitmentofimmunecellstotumorsisanimportantindicatoroftheeffectivenessofanticancerimmunetherapies.Thisrecruitmentcanalsobeusedtoobservetumor-inducedimmunesuppression.Theinteractionsbetweencellsoftheimmunesystemandtumortargetsinthecontextofthetumormicroenvironmentcanbefollowedbyintravitalmicroscopy(56).OneoftheimmunecellsthatiscloselyconnectedtoprogressionofthevarioustypesofcancersistheregulatoryTcell,theactionofwhichisbasedontheimmunosuppressivefunctionoftheimmuneresponse(57).Forexample,Bauerandcolleaguesusedtwo-photonmicroscopytoinvestigatethepro-tumorroleoftumor-experiencedregulatoryTcellinteractionswithdendriticcells.TransgenicmiceexpressingenhancedGFP(eGFP)inallTcellsandmCherryinantigen-presentingcellswereused.TheirstudyshowedthatregulatoryTcellinteractionswithdendriticcellsintumor-draininglymphnodescausedthedeathofdendriticcells(58).Intravitalmicroscopyisavaluabletoolalsoforassessingthedynamicchangesinthetumorvasculatureandfollowingthetranscriptionaltargetingofgeneexpressioninvarioustissues(59,60). NewMouseModelsforCancerResearch Newmousemodelshaveemergedforresearchinpreclinicaloncology,especiallywithinthelastdecadewiththegreatadvancementsinmolecularbiologyaswellasgenomictechnologyandengineering.OnenoveltywastheproductionofnGEMM,whichshowedgreatpromiseinproducingtransgenicmiceatalowcostwithlesstime-consumingprocedures.OthernoveltiesarealternativeDNAmodificationtechniques,whicharemoreefficientforthefasterandcheapergenerationofnewtransgenicmice.AllthedifferentalternativemodificationsofDNAcanbeusedtoproducenewtypesoftransgenicmiceorfurthermodifyconventionalmodels,asdescribedinFigure1.Furthermore,apartfromtransgenicmice,newmodelsusedincancerresearchhaveemerged,suchashumanizedmice,whichhavere-establishedbordersintumormicroenvironmentstudies.Humanizedmiceimplementedwithpatient-derivedxenografts(PDX)canelucidatetheinteractionbetweenthehumantumorandhumanimmunesystemaspartofthetumormicroenvironmentinamousemodel. Non-GermlineGeneticallyEngineeredMouseModels Non-germlinegeneticallyengineeredmousemodelsarecharacterizedasmousemodelscarryinggeneticallyengineeredallelesinsomaticcellsbutnotingermlinecells(22,61).Acomprehensivereviewoftheiradvantagesandlimitationsisprovidedelsewhere(19),andonlyabriefsummaryisgivenherein.ThenGEMMareproducedbytwomajorapproaches:bygeneratingchimericortransplantationmodels.Non-germlinechimericmicecanbeaby-productoftraditionalknockouttechnology(Figure1C),presentingchimericmicethatdonotcarrymodifiedEScellsinthegermlinelineage(62).ChimericmousemodelsforcancerresearchcanalsobeproducedonlyforthepurposeofgeneratingnGEMMsbyinjectinggeneticallyengineered,cancerpredisposed,EScellsintoblastocystsfromachosengeneticbackgroundtodevelopcancer-pronechimericmiceinsomatictissues(63).Asrecipientblastocystsusuallyhaveawild-typegeneticbackground,andnoteverycellinthebodyishencegeneticallymodified,thissituationbettermodelscarcinogenesisinhumans.LargebanksofgeneticallymodifiedmouseEScellsinalargeproportionofgeneshavealreadybeenestablishedingenome-widemutagenesisprogramssuchasEuropeanConditionalKnockoutMouseMutagenesis,NorthAmericanConditionalKnockoutMouseMutagenesis,theUSA-NIHKnockoutMouseProject,andtheEuropeanMouseDiseaseClinicprojects,andtheyarealsocommerciallyavailable(e.g.,https://www.jax.org).Hence,EScellsobtainedfromtheseresourcecenterscanbeimmediatelyusedtogeneratetailorednGEMMs.Ascarcinogenesisisspatiallyandtemporallyrestricted,tissuesordevelopmentaltimespecificitycanbeaccomplishedbyapplyinginductionreagentslocallyorinatime-restrictedmanner.OnelimitationofchimericnGEMMsistheincreasedvariabilityrelatedtotumorigenesisbetweenindividualchimericmiceandthatEScellscanpopulatedifferentcelllineagesandhencedifferenttargetorgans,whichcanproduceheterogeneitybetweenindividualmice(49,64).Additionally,someofthetargetcelllineagescannotbeefficientlyorcannotatallbepopulatedbyEScells. Intransplantationmodels,thetransplantedtissuecanderiveeitherfromgeneticallyengineereddonormicethatcanhaveapredisposingcancermutationorfrommouseorhumancellsthathavebeenpreviouslyengineeredexvivo(mouse-to-mouse;human-to-mousemodelsofnGEMM)(19).Transplantationsystemshavefirstandmostlycommonlybeenadaptedtostudyhematopoieticcarcinogenesis.Here,hematopoieticstemandprogenitorcellsarederivedfrombonemarroworfetalliverandtransplantedbysimpleintravenousinjectionintolethallyirradiatedrecipientmice(65).Irradiationmodelsofhigh-dosechemotherapyinhumansalsocreateawindowforsuccessfulengraftmentoftransplantedmodifiedhematopoieticstemandprogenitorcellsintonGEMMs(66,67).Takentogether,nGEMMsareverypotentfortheiruseincancerresearch,withgreatvalueintestingnewtherapeutics.OneofthegreatestadvantagescomparedtotraditionalGEMMisthefastergenerationofnewtransgenicmiceandimprovementsinmodelingthetumormicroenvironment,whichismoresimilartothesituationinhumancarcinogenesis. AlternativeDNAModificationTechniques Fine-tunemodelingofmousecancermodelscanbeperformedusingseveralalternativemethodsthathavebeendevelopedforfasterandmorereliabletestingofgenesfortheironcogenicpotential.Themostcommonlyusedmethodsaretransposon-basedinsertionalmutagenesis,RNAinterference(RNAi),andengineerednucleases(68). Transposon-BasedInsertionalMutagenesis TransposonsareDNAsequenceswiththeabilitytomovefromonelocationofthegenometoanother.Twogroupsoftransposonsareknown:retrotransposonsandDNAtransposons.Retrotransposons,becauseoftheirlowintegrationefficiency,theintegrationofincompleteretrotranscribedelements,andtheconcomitantinductionofchromosomalaberrations,arerarelyusedfortheproductionoftransgenicmice(69).Incontrast,DNAtransposonshaveshowngreatpromiseintransposon-mediatedinsertionalmutagenesis.Mutagenesisreliesonatransposaseenzyme,whichdistinguishesspecificDNAsequencesand“cuts”theDNAbetweenthem.TheexcisedDNAisthenreintegratedatanothersiteinthegenome(70)(Figure2).Transposon-basedinsertionalmutagenesiscanhencebeusedingeneticscreenstoidentifynovelcancer-causinggenes,suchasoncogenesortumor-suppressorgenes(71).Thetwomosteffectivetransposonsaredescribed:SleepingBeautyandPiggyBac. FIGURE2 Figure2.Mechanismoftransposon-basedinsertionalmutagenesis.Thetransposonsystemiscomposedofatransposon(targetedsequenceofDNA)andanenzyme(transposase).Thetransposasebindstotheappropriatesitealongthetransposonandexcisesthetransposon.ItthenpastesthetransposonatanappropriatelocationinanotherDNAsequence,TAsiteinthecaseofSleepingBeauty,andaTTAAsiteinthecaseofPiggyBac. SleepingBeauty TheimportantelementsofSleepingBeautyarethetransposase,whichisanenzymeusedforthemobilizationofDNA,andthetransposon,whichisamobilizedsequenceofDNA(72).ThemechanismofSleepingBeautyreliesonacut-and-pastemode,andwhenthetransposaseexcisesatransposon,itleavesbehindathree-basefootprint.Then,thetransposoncanmobilizetoanylocationinthegenomewhereaTAdinucleotideispresent.Therearemorethan300millionTAsitesinthegenome.TheTAinsertionsiteisduplicatedduringtheprocessoftransposonintegration(70,73).Thetransposoncancarryanysequenceofchoice,butthetranspositionefficiencydecreaseswithanincreasedsizeofthesequence(70).Thesesequencescanbemutagenicelements,whichcanbeintendedtoimitatethosepresentinretroviruses.TheSleepingBeautytransposonscanbeusedfortheinductionofloss-of-functionmutationsaswellasgain-of-functionmutations(74).Duetothecut-and-pastemode,thereisonlya40–50%possibilitythatreintegrationoftheexcisedtransposonwilloccurintothegenome.Additionally,becausethenumberoftransposonsintegratedinthegenomedecreasesovertime,alargenumberoftransposableelementsarerequired(75).SleepingBeautycanbeusedforthediscoveryofcandidatecancergenesandtosearchforthedriversofmultiplecancertypes.Becauseofthesescreens,severalcancer-promotingmutationscandidatehavealreadybeenfound,whichcanbeusedinthedevelopmentofnewmousemodelsthatmayproveusefulfortherapeutictesting.Toidentifycandidatedriversofcolorectalcarcinoma,transposon-basedscreensareusefulbecausecancer-promotingmutationsarecausedbytransposoninsertioneventsratherthangenome-wideinstability.ColorectalcarcinomahasbeenmodeledusingmicecarryingthemutagenicT2/Onc2SBtransposons,conditionalRosa26-lsl-SB11transposase,andvillin-Cretoactivatetranspositionspecificallyingastrointestinaltractepithelialcells.Usingthistechnique,intraepithelialtumors,adenomas,andadenocarcinomasinthesmallandlargeintestinesweregenerated.Moreover,analysesofthetransposoninsertionsiteofthesetumorsidentified77candidatecolorectalcarcinomagenes,60ofwhichareknowntobealteredordysregulatedinhumancolorectalcarcinoma(76).Furthermore,SleepingBeautycanalsobeusedtoinducecancerinatissueofinterestbycombiningitwiththeCrerecombinaseinduciblesystem.ByemployingCreexpressionunderthecontrolofanalbuminenhancerorpromotorsequence,whichisspecificforliver,SleepingBeautytranspositionwaslimitedtotheliver.Thissystemwasusedtoscreenforhepatocellularcarcinomaassociatedgenes.Newgenespotentiallyinvolvedincarcinogenesis,suchasUBE2H,werediscovered,andtherefore,thismodifiedsystemwasintroducedinthesearchfornewpossiblecandidategenes(77). PiggyBac PiggyBactransposonsaretheonlyefficientalternativetoSleepingBeautyforcancergenediscovery.ComparedwithSleepingBeauty,PiggyBaccancarrylargercargos(uptoseveralhundredkilobases).Thesecargosareinsertedwithhighertranspositionactivityintomammaliangenomes.Additionally,thePiggyBacsystemrequiresaTTAAinsertionsiteinsteadofTA,andafterthetransposaseexcisesatransposon,itdoesnotleaveanyfootprint,incontrasttoSleepingBeauty.ThisimpreciseexcisionofPiggyBaccanleadtodamageatthemobilizationsite,therebycreatinglossorgain-of-functionalleles(70,78).ThePiggyBactransposonscanalsobeusedtogeneratetransgenicrodentsexpressingareporterfluorescentproteinindifferentorgans.Recently,transgenicratscarryingeithertheRFPgeneortheeGFPgeneweregeneratedbyinjectingpronucleiwithPiggyBacplasmids.NotonlydidthetransgenicratsexpresstheRFPandeGFPgeneinmanyorgans,buttheyalsohadthecapabilitytotransmitthereportergenetothenextgenerationthroughintegrationintothegermlinelineage(79). RNAInterference RNAinterferenceinmicerepresentsanalternativetoknockoutmice,or,moreaccurately,aknockdownmouse.Namely,knockdownbyRNAidoesnotgenerateacompletelyloss-of-functionallele(80).Silencing,orbetter,downregulatinggeneexpressionofatargetgenebysmallinterferingRNA(siRNA)hasbeenmainlyusedtostudygenefunction(81).Itwasusedforsilencingestrogenreceptoralpha(ESR1),wherestableknockdownsuppressedtheproliferationandenhancedapoptosisofbreastcancercells(82),orforsilencingtransketolase(TKT),whichaffectscellproliferationandmigrationaswellasinteractionswithothermetabolism-associatedgenesinlungcancercells(83).However,theknockdowneffectofsiRNAisonlytransientduetotheshorthalf-lifeofsiRNAmolecules.Toachieveamoresustainedgene-silencingeffect,plasmidsencodingshorthairpinRNAs(shRNA)canbeused.RNAibyshRNAspermitsreversiblesilencingofgeneexpressionwithoutalteringthegenome.ToincreasetheexpressionoftheshRNA,thetargetingvectorofinterestcanbeinsertedintotheRosa26locusbytherecombinationofasite-specificrecombinaseinEScells(developedusingatechniquedescribedinFigure1B). EngineeredNucleases Thusfar,threekindsofengineerednucleaseshavebeendevelopedandtestedforDNAmodulation:zinc-fingernuclease(ZNF),transcriptionactivator-likeeffector(TALEN)nuclease,andthelatestclusteredregularlyinterspacedshortpalindromicrepeat(CRISPR)/-associated(Cas9)system(84). Briefly,ZNFsandTALENsareproducedbycombiningaDNA-bindingdomainwithaDNA-cleavagedomain.Thesedomainscanbeengineeredtoactasasite-specificnuclease,cuttingDNAatstrictlydefinedsites,whichenableszinc-fingerorTALENnucleasestotargetuniquesequenceswithincomplexgenomes.ThetargetingefficiencyoftheZNFsystemreaches68%(85),andZNF-mediatedgene-targetingexperimentsarearelativelyefficientmeansforgeneratingnon-homologousend-joining(NHEJ)-mediatedknockoutmice(86).UsingtheTALENmethodtoproduceknockoutmiceisefficientin49–77%ofcases(87),whichcanbeincreasedwithagreaterconcentrationofTALENmRNA.Thismethodhasbeenprimarilyusedtoincreasetheefficiencyofgenetargeting,andcomparedtoZNFs,TALENsyieldhighermutationefficienciesandsurvivalrates. However,theuseofZNFsandTALENsislimitedbecauseconstructionoftheproteindomainsforeachparticulargenomelocusiscomplexandexpensive.Additionally,singlenucleotidesubstitutionsorinappropriateinteractionbetweendomainscancauseinaccuratecleavageofthetargetDNA(84).Furthermore,thetargetingefficiencymaybevariableandmuchlowerthanreportedabove.However,amajordrawbackisthatsimultaneousgenetargetinginmultiplegenesishindered,preventingstudiesofoncologicalphenotypeswhereinmultiplemutationsarerequired,inanalysesofgenefamilymemberswithredundantfunctionsorincasesofcancersinwhichgene–geneinteractionsexist. CRISPR/Cas9System ThesimplestandthemosteffectiveengineerednucleasesystemtogeneratetransgenicmiceistheCRISPR/Cas9system(88).ComparedwithZFNsandTALENs,theCRISPR/Cas9-mediatedgenomeeditingismoreefficient,andthedesign,constructionofreagents,aswellasdeliveryareeasier.Additionally,targetedmutationsinmultiplegenes(so-calledmultiplexgenomeengineering)arepossiblewiththeCRISPR/Cas9system.ThissystemconsistsofaCas9nuclease,whichcanbedirectedtoanygenomiclocusbyanappropriatesingleguideRNA(sgRNA).Untilnow,threemaintypesofCas9variantshavebeendevelopedthatdifferintheirmechanismsofaction.Thefirstsystemtobeadaptedformousetransgenesiswasthewild-typeCas9proteinfromthetypeIICRISPRsystemofStreptococcuspyogenes,whichfunctionsviaanassociationwiththesgRNAwitharelativelyshortrecognitionsequence(~20nt)(89).Fordouble-strandcleavage,thissystemrequirestheprotospacer-adjacentmotif(PAM),whichis“NGG”or“NAG”forS.pyogenesCas9atthe3′endofthetargetsequence.Recently,newformsofCas9enzymeshavealsobeendevelopedthatcanbindtoalternativePAMsitesandtherebyextendtherangeofutilityofCas9(90).Oncethedouble-strandedbreaksoccur,itcanberepairedbyNHEJorbyhomology-directedrepair(HDR)(91)(Figure3).NHEJ-mediatedrepairfrequentlyresultsinshortinsertionsordeletionsthatgenerateloss-of-functionmutations. FIGURE3 Figure3.MechanismofCRISPR/Cas9genemodulation.AsingleguideRNAdirectsCas9nucleasetoagenomiclocus,whereitcutsthetargetsequenceinthepresenceofprotospacer-adjacentmotif.Theresultingdouble-strandedbreaksstimulatesDNArepair,whichcanoccurvianon-homologousend-joiningorhomology-directedrepair-mediatedrepair. ThesecondCas9variantwasdeveloped(92)toincreasetheefficiencyofHDR,allowinginsertionsorreplacementsofspecificnucleotides.AmutantformCas9protein(Cas9D10A,callednickase)wasdevelopedthatcleavesonlyoneDNAstrand,downregulatingtheactivationofNHEJ.WhenahomologousrepairDNAtemplatewithaspecificmutationorsequencetobeintroducedisprovidedinthemixtureofthesgRNAandCas9D10Amutation,itcanserveasatemplatetorepairthelesion.Thisactivatesthehigh-fidelityHDRpathwayandhenceoffersthepossibilitytogenerateallelereplacementsandotherspecificmodificationsinthemousegenomethatwereessentiallyimpossiblewiththeclassictransgenesismethodsdescribedinFigure1. ThethirdCas9variantistheso-called“dead”Cas9ornuclease-deficientCas9(dCas9)(93),inwhichcertainmutationswereintroducedtoinactivatethecleavageactivitybutretaintheDNA-bindingactivity.ThisvariantwasdevelopedtobeabletotargetanyregionofthegenomewithoutcleavageandbyfusingdCas9withvariousactivatororrepressordomains,toup-ordownregulatethetranscriptionoftargetgenes.AnadditionalapplicationofthedCas9systemwasdevelopedbyChenandHuang(94).ByfusingdCas9toeGFP,theydevelopedavisualizationtoolanddemonstratedthattheycouldvisualizeseveraldynamicprocesses,suchastelomeredynamicsduringelongationordisruption,subnuclearlocalizationofcertainloci,anddynamicbehaviorduringmitosisinlivinghumancells. ApplicationoftheCRISPR/Cas9SysteminOncology SeveralsuccessfulapplicationsoftheCRISPR/Cas9systemincancerresearchhavebeenpublishedbyusingoneoftheaforementionedthreesystemsorbycombiningtheclassictransgenicmodelsdescribedinFigure1withCRISPR/Cas9togenerategermlineornGEMMmousecancermodels(88).SomeearlysuccessfulattemptstodevelopnewinvivocancermodelsincludeanewpancreaticcancermodelcombiningviralvectordeliveryandCRISPR/Cas9-mediatedsomaticgenomeediting(95),andalungcancerknock-inmodel(96).ThelatterwasdevelopedbycombiningaCre-dependentCas9mousemodelwithsgRNAdelivery,whichgeneratedloss-of-functionmutationsinp53andLkb1,aswellasnucleotidereplacementleadingtoanoncogenicK-rasG12Dmutationthatcauseslungadenocarcinoma.Aconditionalliver-specificmutationincancergeneswasdevelopedbyXueetal.(97),whereasthedevelopmentofnovelbraintumormousemodelswasreportedbyZuckermannetal.(98).AnimportantstepforwardinnewmodelsincancerresearchwasdemonstratedbyMaddaloetal.(99),whousedCRISPR/Cas9-mediatedinvivosomaticgenomeeditingtoengineerchromosomalrearrangements.Thisclassofmutationsplaysanimportantroleincarcinogenesis,butitisverydifficult,ifnotimpossible,todevelopusingclassicaltransgenesisapproaches(Figure1).Authorshaveusedviral-mediateddeliveryoftheEml4–AlkfusiongenebytheCRISPR/Cas9systemtosomaticcellsofadultanimals,whichmodelsaninversiononchromosome2:inv(2)(p21p23)thatoccursinhumans.ExpressionoftheEml4–AlkfusiongeneinthismodelresultsinpathologicalandmolecularcharacteristicsoftypicalALK+humannon-smallcelllungcancers(NSCLC).Moreover,thismousemodelrespondspositivelytoALKinhibitors.Similarly,usingasomaticCRISPR/Cas9approach,Cooketal.(100)demonstratedinanexvivoandinvivostudythatachromosomalrearrangementresultinginBcan–Ntrk1fusioncreatesapotentdriverforglioblastomadevelopment. TheadaptabilityoftheCRISPR/Cas9systemtothescientificquestionandarelativelyeasywaytoscaleuptheexperimentaldesignhasalreadyledtohigh-throughputinvivoscreenstocatalogfunctionaltumorsuppressors.OnesuchcomprehensivestudybyWangetal.(101)mappedfunctionalcancergenomevariantsoftumorsuppressorsinthemouseliverofthewild-type,immunocompetentstrain.ByinjectingAAVpoolscontainingalarge(278)sgRNAlibrarydirectedtowardknownandthemostfrequentlymutatedtumor-suppressorgenesintoRosa-Cas9-eGFPknock-inmice,theywereabletogenerateamutationalatlasoflivertumors.AllthemicethatreceivedthisAAV-sgRNAoftumor-suppressorsgRNAdevelopedlivercanceranddiedwithin4months,demonstratingthevalidityandextremelyhighefficiencyofthisscreeningapproach.Therefore,AAV-mediatedCRISPR-Cas9screensprovideapowerfulhigh-throughputtoolformappingfunctionalcancertumorsuppressorsinvarioustissuesinfullyimmunocompetentmice. Studiesusingwild-typeCas9ornickasemutationCas9varianthavethusfarbeenmostfrequentlyusedinmousecancermodeldevelopment.However,applicationofthedCas9systeminwhichnogenomemodificationsareproducedbutaneffectontheexpressionoftargetgenesisobservedhavealsostartedtoemergeininvivomodelsofcancer.AgoodexampleofthistypeofresearchhasbeendescribedinBraunetal.(102),whoaimedtoexaminetheeffectoftheupregulationofMgmtusingdCas9proteinfusedtoafourfoldrepeatoftheVP16transcriptionalactivator(VP64)incombinationwithsgRNAstargetingupstreamregulatoryregions(103).ThistargetgenewaschosenbecauseitisknowntodetoxifyDNAlesionscausedbythechemotherapeuticagenttemozolomide.MurineacuteB-celllymphoblasticleukemiacellswerefirstinfectedwithacombinationofdCas9-VP64andsgRNAsandtransplantedintowild-typefullyimmunocompetentC57BL6/Jmice.Positiveresultswereobtained,asupregulationofMgmtwasachieved,andthemicerespondedtotemozolomide.ThesefindingsdemonstratedthatthedCas9-basedsystemcouldbesuccessfullyusedtoaffectgeneexpressiononlyandtomodeloncologicalgeneticmodificationsduringtreatmentrelapseinvivo. Furthermore,thesimultaneousinjectionofCas9mRNAandsgRNAintothecytoplasmofzygoteshasbeenshowntoefficientlyandreliablygenerateknockoutmicewiththehighesttargetingefficiency(67–100%)ofallengineerednucleases(84).Beyondthedevelopmentofnoveltransgenicmice,CRISPR/Cas9canalsobeusedtorefineexistingmodelsofcancerbyreengineeringEScelllinesfromwell-knowntransgenicmicetoharboradditionalconstitutiveorconditionalmutantallelesofoncogenesandtumor-suppressorgenes(104).Therefore,CRISPR/Cas9representsanefficientmethodforgeneratingtransgenicmiceduetoitssimplicity,cost-effectiveness,highefficiency,andlowfetaltoxicityevenatrelativelyhighdosesofCas9mRNAandsgRNA(105). HumanizedMouseXenograftModels Patient-derivedxenograft(PDX)modelshavebeenextensivelyusedinstudiesofvarioussolidandhematologicmalignancies,suchasbreastcancer,colorectalcancer,pancreaticcancer,chroniclymphocyticleukemia,andlargeBcelllymphoma(106,107).PDXmodelsareusedfortheassessmentofhumantumorbiology,identificationoftherapeutictargets,andareanimportantmodelforpreclinicaltestingofnewdrugsforvariouscancers.PDXmodelsareestablishedbytheimplantationofcancercellsortissuesfrompatientprimarytumorsintoimmunodeficientmice.Severaltypesofstandardimmunodeficientmiceexist,suchasathymicnude,SCID,NOD-SCIDandrecombination-activatinggene2(Rag2)knockoutmice(108).However,thesemousemodelsareusuallyusedtoestablishaxenograftcancercelllineortogrowtransplantabletumorxenografts,andtheyareunabletogrowprimarycancercellsortissues.Toaccomplishthisgoal,greaterimmunodeficiencyisrequired,whichisprovidedbythegenerationofNOD/SCIDmicewithIL2rgmutations(NSG)thatareabletoengraftalmostalltypesofcancerduetotheirenhancedimmunodeficiency(109).Toimplantpatient-derivedtumorsintoimmunodeficientmice,smallfragmentsoftumors,cellsuspensionsderivedfrombloodorfromthedigestionoftumorsintosingle-cellsuspensionsareused.Theimplantationcanbeperformedheterotopicallyororthotopically.Heterotopicimplantation,forexample,subcutaneously,hasadvantagesoverorthotopicimplantationduetothesimplicityofthemethodandmoreconvenientmeasurementoftumorsize.SubcutaneousandintravenousPDXmodelsaremostwidelyusedincancerresearchforsolidtumorsandleukemias.Incontrast,ifthemainaimoftheresearchismetastasesofcertaincancertypes,thanorthotopicmodelsaresuperiorbecauseorthotopicimplantationintohosttissuescanproducemetastasesviathenormalprocessofcancerprogression(110). Duetorecentadvancesinimmunotherapyilluminatingtheimportanceoftheimmuneresponseintumorprogressionandtreatment,newPDXmodelsarenecessary,namelyPDXmodelstogetherwiththehumanimmunesystem,inwhichtheinteractionbetweenhumancancersandthehumanimmunesystemcanbeinvestigated,aswellaspotentialantitumorimmunotherapies(107).Severalmethodscanbeusedtoproducetheseso-calledhumanizedmousemodels.Onesuchmodelcanbeproducedbythetransplantationoftotalperipheralbloodortumor-infiltratinglymphocytesintoimmunodeficientmice.However,thesemethodsareverylimitedincancerresearchbecausetheycauseseveregraft-versus-hostdisease(111).Therefore,anothermethodhasbeenusedtoproducehumanizedmousemodelsthroughthetransplantationofCD34+humanhematopoieticstemcells(HSCs)orprecursorcellsisolatedfromumbilicalcordblood,bonemarrowandperipheralblood,asshowninFigure4.TransplantationofHSCsgivesrisetovariouslineagesofhumanbloodcellsinmice(112). FIGURE4 Figure4.SchematicillustrationofhumanizedPDXmousemodelproduction.CD34+humanhematopoieticstemcells,whichareisolatedfromumbilicalcordblood,aretransplantedintoNSGmice.Thisprocessleadstothedevelopmentofhumanhematopoieticandimmunesystems.PDXofvarioustumorscanthenbeimplantedforfurtherresearch. Thesehumanizedmodelscanbeusedtoinvestigatetheefficacyandmechanismofcancerimmunotherapy,suchasprogrammedcelldeathprotein1(PD-1)-targetedimmunotherapy.Wangetal.(113)describedthedevelopmentofhumanizedNSG(huNSG)micebytransplantationofhuman(h)CD34+hematopoieticprogenitorandstemcells,whichledtothedevelopmentofhumanhematopoieticandimmunesystems.Subsequently,theyimplantedthePDXofNSCLC,sarcoma,bladdercancer,andtriple-negativebreastcancerintosuchhumanizedmice.TheydiscoveredthattumorgrowthcurvesweresimilarinhuNSGincomparisontonon-humanimmunecell-engraftedNSGmice.Treatmentwiththecheckpointinhibitorpembrolizumab,anantibodythattargetsPD-1,causedsignificantgrowthinhibitionofPDXtumorsinhuNSG,butnotNSGmice.Theseresultssuggestthattumor-bearinghuNSGmicecouldrepresentanimportantnewmodelforpreclinicalimmunotherapyresearch.AsimilarresultwasobtainedbyPanetal,(114),whoinvestigatedtheantitumoreffectivenessofpembrolizumabinhumanbladdercancerPDXinhuNSGmice.TheyobservedthattreatmentwithpembrolizumabinhibitedtumorgrowthanddecreasedthenumbersofCD4+PD1+andCD8+PD1+cellsinperipheralbloodandincreasedthenumbersofCD45+andCD8+cellsinPDXs.OnelimitationofNSGmiceisthatdespiteengraftmentwithhumanCD34+cells,thesemicewillacquireonlypartiallyfullymaturehumanbloodcellsduetoincompatibilitybetweenthemouseandhumancytokinesnecessaryforbloodcelldevelopment.Recentmodelsaimtoachievethecombinationoftransgenicorknock-inmousemodelsexpressinghumancytokinestogetherwithNSGandCD34+celltransplantationtoimproveengraftment(115). Furthermore,recentstudieshavedemonstratedthatthemicrobialecosystemhasamajorimpactonthelocalanddistantimmuneresponseandthattheefficacyofimmunetherapieswithcheckpointinhibitors,suchaspembrolizumab,canbediminishedbytheuseofantibioticsandenhancedinthepresenceofspecificgutmicrobes.Tofullyevaluatetheinterplaybetweenimmunotherapiesandthemicrobiota,newmousemodelsareemerging,suchasspecificpathogen-freemicewithdefinedcommensalbacteriaorpreconditionedwithantibiotics,orgerm-freemicelackingcommensalbacteria(116).CommensalbacteriasuchasBifidobacteriaspp.andAkkermansiamuciniphilacanincreasetheefficiencyofanti-programmedcelldeathprotein1ligand(PD-L1)-basedimmunotherapyagainstepithelialtumorsbyimprovingtumorcontrol(117–119).Additionally,acorrelationbetweentheuseofanotherimmunecheckpointinhibitor,ipilimumab(antiCTLA-4antibody),andcolonizationbyBacteroidaleswasobserved.TheefficacyofCTLA-4blockadewasimprovedbythemicrobiotacompositionofBacteroidales,whichaffectsinterleukin12-dependentTh1immuneresponses,thusenablingbettertumorcontrolinmicewhilesparingintestinalintegrity(120).OnelimitationofthesemousemodelswithengraftedhumanmicrobiotaisthatthesemicearelikelyunabletosupportcolonizationbyallcommensalsofthehumanGItract;therefore,itmaybesufficienttofocusonbacteriathatsuccessfullycolonizebothhumansandmice. CurrentDirectionsinTransgenicMouseCancerModels Themousecancermodelsdiscussedintheprevioussectionsclearlyshowagreatimpactofthesemodelsonthestudyofbasicmechanismsofcarcinogenesis,aswellastheevaluationordevelopmentoftherapiesthatarepotentiallyapplicableinhumanoncology.However,bothtraditionaltransgenicmodelsandnewopportunitiesofferedbyCRISPR/Cas9providegreatpromiseinevenmoreefficientandtranslatablemousemodelsforcancerresearchinthefuture.Inthissection,wediscussselectedfieldsinwhichwepredictmajordevelopmentsinthenearfuture:personalizinghumanizedmice,replicatingspecifichumanmutationsinmousemodels,analyzingandmanipulatingthe“cancer”epigenome,andprospectsintheuseofmousemodelsforgenetherapyapplicationsinhumans. PersonalizingHumanizedMice Humanizedmicehaveshowngreatpotentialinpreclinicaloncologystudies.Tofurtherincreasethepotentialofthesemodels,thereisanecessityfortheimmunesysteminhumanizedmicetobecompatiblewithbothitshostenvironmentandwiththeimplantedtumortissuetoaccuratelymodelthepatient’simmuneresponseduringtreatment.Tissueincompatibilityofhumanizedmicethatareengraftedwithanimmunesystemfromonepersonandimplantedwiththetumorofanothercouldbethereasonfortheimmuneresponseobservedinhumanizedmice,whichisthusnotrelatedtothespecifictreatmentappliedtothemice.WhenhumanizedmiceareproducedfromtheengraftmentofCD34+cells,someofthematurexenoreactiveTcellsarealsointroducedintothesemice.TheseTcellsdifferentiatewithintheengraftedbonemarrow,maturewithinthemouseandseemtodisplaysomexenoreactivetendencies(108).However,becausethetransplantedhumanimmunesystemisweakened,itpreventscompleterejectionofthexenograft.OnepossiblesolutiontothisproblemcouldbetheproductionofahumanizedxenograftmodelinwhichtheCD34+cellsandimplementedtumortissuearederivedfromthesamedonor.Kleinetal.producedhumanizedmiceusingCD34+bloodcellsisolatedfrombiopsiedbonemarrowofbreast,lung,prostate,oresophagealcancerpatient,raisingthepossibilityofindividualizedanalysesofantitumorTcellresponses(121).Moreover,anewmelanomaPDXmodelhasbeendesignedwhereintumorcellsandtumor-infiltratingTcellsfromthesamepatientaretransplantedsequentiallyinNOG/NSGknockoutmice.Thismodelwasdevelopedtostudythemostadvancedandmostpromisingcurrentanticancertherapies,immunecheckpointinhibitorsandadoptivecelltransferofautologoustumor-infiltratingTcellsthathavedemonstratedcompletedurableresponsesinasubpopulationofpatientswithadvancedmelanoma(122). ReplicatingSpecificHumanCancerMutationsinMouseModels TheconventionalmousemodelsdescribedinFigure1willcontinuetobeusedincancerresearchbothontheirownandincombinationwithotherapproachessuchastransplantationmodelsandhumanizedmice.However,asalludedpreviously,allthreemajortraditionaltransgenesistechniquessufferduetoaninabilitytoefficientlydeveloppreciseallelereplacementsorinsertions.Initially,CRISPR/Cas9-mediatedmutagenesiswashighlyeffectiveforgeneratingloss-of-functionmodelsbutnotpreciseallelereplacementsorgain-of-functionmutations,whicharemostfrequentincancer.However,recentimprovementsintheCRISPR/Cas9systemhaveimmenselyincreasedtheefficiencyofHDR[e.g.,Gutschneretal.(123);Komoretal.(124)]andhencetheabilitytoengineerprecisemutationsatanysiteinthegenome.Somesuccessfulallelereplacementsinthecancerresearchfieldhavealsobeenachievedinvitro.Forexample,Burgessetal.(125)developedthehomozygousreplacementoftheoncogenicG13DK-RASmutationinahumancolorectalcancercellline,whichrenderedthemsensitivetodrugtreatment.OnemajornoveltyoftheCRISPR/Cas9systemistheabilitytosimultaneouslygeneratemultiplemutations.OnesuchsuccessfulattemptwasrelayedinastudybyWaltonetal.,whomanagedtogeneratetriplegenemutationsthatmadecellsdeficientinTrp53,Brca2,andPtengenes(126).Novelgenefusionmutationsarefrequentlyfoundinhumancancers.TomodelonesuchfusioninmicelinkingDnajb1–Prkacagenesintoonetranscript,Engelholmetal.(127)employedCRISPR/Cas9methodtopreciselydeletearegioninmicethatissyngeneictothehumanregiononchromosome8torecreateaDnajb1–Prkacafusion.Theydemonstratedthatthisfusionistheonlydrivertoinducehepatocellularcarcinoma,withseveralfeaturesresemblinghumanlivercancer. Apartfromprecisemutationsencompassingonenucleotideorsmallergenomicsegments,asdescribedabove,CRISPR/Cas9technologyalsooffersopportunitiestogeneratelargechromosomalaberrations.Recently,studieshavebeenpublishedwiththeaimtoimprovetheefficiencyofgeneratingchromosomalrearrangements.Onesuchstrategy,namedCRISprMEdiatedREarrangementstrategy(128),hasprovenveryefficientinproducingdesiredrearrangementsfromonesingleexperiment.Targetedlargedeletions(upto24.4Mb),duplications,andinversionsinrodentmodelsweredevelopedusingthisapproach,whichwillprobablysoonbeusedincancerresearchtomodelchromosomalaberrationsinvolvedintumorbiology. Cancerisalsocharacterizedbymultipleepigeneticchangesthatcandrivecarcinogenesisandconferresistancetotreatment.Epigenomeediting,especiallybytheCRISPR/Cas9system,nowallowsanalysesofpreciseepigeneticmodificationsandtheireffectsoncancerdevelopmentandtherapy.Onegreatchallengeaheadwillbetoachievethereversionofepigeneticmodifications,includingDNAmethylationandothermechanisms(e.g.,histoneacetylation)atprecisesitesandensurethatsuchaninterventionismitoticallyheritable.Somerecentstudiesincelllineshavedemonstratedthatselectiveepigeneticchanges(e.g.,DNAmethylation)canbeachievedwiththeexpectedoutcomeontheexpressionoftargetgenes(129).ApartfromDNAmethylation,posttranslationalmodificationsofproteins,suchashistoneacetylation,alsopresentanimportantepigeneticmechanismofgeneexpressiondisruptionthatcanleadtocarcinogenesis.InarecentstudybyShrimpetal.(130),dCas9fusedtoanactivator,p300,tocontroltheexpressionoflysineacetyltransferases(KATs)wasapplied.ThispioneeringstudydemonstratedthepotentialofthedCas9-p300systemforstudyinggeneexpressionmechanismsinwhichacetylationplaysacausalrole,whichiscertainlythecaseincancerbiology.Furtherdevelopmentsinthisareaofresearchmayleadtothedevelopmentofmethodsforthespatiotemporalcontrolofacetylationatspecificloci,whichinturncouldleadtotherapeuticeffects.TheabilityofthedCas9-effectorsystemtoactivateorrepressendogenousgeneexpressionalsoprovidesanewanduniqueopportunitytofurtherexaminecancer-associatedcisortransactingregulatorynon-codingRNAs.Thus,recentdevelopmentsinCRISPR/Cas9technologydemonstrategreatpromiseforfutureuseandapplicationintransgenicmousemodelsforstudyingcancerbiology. DeliveryMethods Intransgenicmousemodels,thedeliveryofcomponentstoinducemutationsortodelivermodifiedcellsinvivostillpresentsamajorchallenge.Abriefreviewofthedeliverymethodsusedincancermousemodelsisprovidedbelow,withafocusontheCRISPR/Cas9system.ThedevelopmentofdeliveryvehiclesforCRISPR/Cas9-mediatedtransgenesis,especiallyinthegenerationofinvivomousecancermodels,hasbeenchallengingbecauseoftherequirementsforthedeliveryofmultiplecomponentsinaspatiallyortemporallycontrolledmanner.Nevertheless,somedeliverymethodshavealreadybeenattemptedinmousemodelsofcancerandvarywidelydependingonthetargetcancertypeorscientificquestionsasked. IntravenousinjectionofCas9-editedhematopoieticstemprogenitorcellshasbeensuccessfullyappliedtomodelmyeloidmalignanciesinmice(131)andinaBurkittlymphomamodel(132).Electroporation-baseddelivery,awidelyusedmethodfortheintroductionofdifferentmolecules(chemotherapeuticdrugsandgeneticmaterial)intodifferenttypesofcellsinvitroandinvivo(133),hasalsobeenusedininvitrocancermodeling,forexample,inmodelingalveolarrhabdomyosarcomainmousemyoblasts(134)aswellasinvivoforhematopoieticcell-basedtherapyofmalignancies(135).Aso-calledhydrodynamictailveininjectionofCRISPR/Cas9componentshasbeenappliedinahigh-throughputmultiplex-mutagenesislivercancerscreen(136).Similarly,inagenome-widescreenoflungcancerinmice,subcutaneousinjectionswereused(137).ForNSCLC,basicepithelialcelltransfectionhasalsobeenusedtotargetgenomicrearrangements(138).TodeveloptransgenicmousemodelsharboringCRISPR/Cas9-inducedmutationsineverycellofthebody,classicalmicroinjectionsintofertilizedeggsorblastocysts(formodifiedEScells)arefrequentlyemployed(139).Recently,somesuccessfulattemptsutilizingtheelectroporationofpronuclearzygoteshavealsobeenreported(140,141).Transfectionwiththepolyethyleneiminereagentincombinationwithelectroporationhasbeenemployedtostudybraintumormodel(98).Viralvector-basedtransfectionshavealsobeenattemptedinvivo.Forexample,AAVdeliveryhasbeenusedtostudylungcarcinogenesisbyapplyingthemintra-tracheallyinvivo(96).Furthermore,lentiviral-basedconstructswereusedinatrialinvolvingapancreaticductaladenocarcinomamousemodel(142). Althoughtheabovereviewofvariousdeliverymethodsthathavealreadybeenattemptedintransgenicmousemodelsofcancerdemonstratessomedegreeofinitialsuccess,severalchallengesremaintobesolved.OnesuchchallengeistoenablethedeliveryofCas9ribonucleoproteincomplexesanddonorDNAinvivotoinducehomology-directedDNArepairandrepaircancer-causingmutations.AveryrecentstudybyLeeetal.(143)usedgoldnanoparticlesconjugatedtoDNAandcomplexedwithcationicendosomaldisruptivepolymers,andtheresultsdemonstratedcorrectionoftheDNAmutationthatcausesDuchennemusculardystrophyinmice.Suchanapproachshouldbeofinterestformousecancermodels,especiallyinheritedformsofdrivermutations.Finally,improvementsindeliverymethodstoincreasespecificityandefficiencyandtominimizeoff-targeteventsandimmuneresponsearenecessarytoensurethevalidityofmousecancermodelsandtoincreasetheirtranslationalpotential. PitfallsandLimitations Aswitheverynoveltechnology,therearepitfallsandlimitationsthatmustbeovercomeusingtheCRISPR/Cas9systeminthefuture.Forexample,inmodelingsmalldeletionsandinsertions,currentCRISPR/Cas9-basedgeneeditingusesNHEJ-mediatedmechanismsthatgeneratesmallindels,butthesequencevariationinthegeneratedallelicseriesisenormous.Whileindelsusuallygenerateloss-of-functionalleles,certainindelscanbein-frameorout-of-frame,generatingtruncatedormodifiedgeneproductswithdifferentphenotypiceffects.Apartfromtheaforementionedloss-of-functionmodels,agreaterchallengeisstillthedevelopmentofprecisecancer-drivermutationsinvivobytheHDRmechanism.Thisapproachcontinuestohaveroomforimprovementstoefficientlygenerategain-of-functionmutationsthatareprevalentincarcinogenesis.Asmentionedearlier,theCRISPR/Cas9systemallowsmultiplexingandhencesequentialmutagenesisofcancergenestomodelloss-orgain-of-functioneventsthatarefrequentlyfoundinhumancancergenomes. Theoff-targeteditingactivityoftheCRISPR/Cas9systempresentsaconcernandpotentiallimitation.ThisactivitycouldaffectthephenotypeofCRISPR/Cas9-generatedmousemutants,suchthatthephenotypeisnotrelatedtotheon-targeteventbutrathersomemodification(s)elsewhereinthegenome.Whilesomestudiesinhumancellsreportarelativelyhighfrequencyofoff-targetevents(144),earlydatainmouseembryossuggestthatCRISPR/Cas9off-targeteventsareveryrare(89,145).Toexamineindetailtheextentofoff-targetevents,next-generationwhole-genomesequencinghasrecentlybeenused.Suchstudiesnowshowthatthelikelihoodofoff-targeteventscanbeminimizedbythecarefuldesignofguideRNAsandselectionofgenomictargetsites(146).Thisresultisfurthersupportedinalarge-scalescreenforoff-targeteventsinCRISPR/Cas9transgenicmiceperformedbySinghetal.(105).ForgRNAsselectedtohavelowoff-targethitscores,90foundermicewerescreenedin56ofthehighest-scoringoff-targetsites,butnocasesofoff-targetmutagenesiswererecorded.Tofurtherminimizetheoff-targetactivityofCas9,whichwillespeciallybeimportantineventualhumantherapy,researchershaveattemptedtomodifytheCas9proteinitself,byusingatruncatedgRNAorbyamethodof“pairednicks”(147,148).DirectuseofrecombinantCas9proteincanalsolowertheoff-targeteditingfrequency,mostlikelybecauseCas9proteindegradesmuchfasteronceitisinthecellthantheplasmidencodingCas9(149).Althoughsomerecentstudiesreportadvancesinminimizingoff-targeteffects(150),bothfuturepreclinicalandespeciallyclinicalapplicationswillrequireessentiallynodetectablegenome-wideoff-targetactivity.Developmentsintheareaofhigh-throughputgenome-widesequencingwillcertainlyaidinallowingtheefficientidentificationofsuchoff-targeteffects(151)andshouldberoutinelyusedinfuturecancermodelstudies. AnotherareathatwillmostlikelygainmoreattentionisthecombinationofconventionalcancermodelswithCRISPR/Cas9toolstoeditgenesandsimultaneouslyaffectgeneexpressionwithoutanygenomeediting.Suchorthogonalapproachesforusingthenucleaseactivity-deficientdCas9-effectorsystemincombinationwiththeeditingCas9-basedsystemshouldsoonbemorefrequentlyappliedinmousemodelsofcancer.Namely,Cas9variantsisolatedfromdifferentbacterialspecies(152,153)ormutatedformsofCas9fromthesamespeciesthatrecognizedifferentPAMsitesnexttothesgRNA-bindingsite(154)arenowavailable.Suchcombinatorialapproachescanbeusedtogeneratemorecomplexmousemodelsofhumancancers,whichiscertainlyacomplexdisease. Conclusion Traditionalmousecancermodelshavealreadycontributedimmenselytowardilluminatingthemechanisticunderpinningsofcarcinogenesisandwillcontinuetobeusedontheirownorincombinationwithmorerecentlydevelopedmodels.Onecriticismregardingtheuseoftraditionalmousetransgenicmodelsliesintheirlimitationswithrespecttothemodeldesignandrelativelyslowtranslationalpotentialformorerapidandimprovedbenefitsforcancerpatients. Inrecentyears,newmousemodelsofhumancancerweredevelopedthatmayovercometheselimitationsbyacceleratingthedetectionofnovelcancergenes,decipheringmechanismsofcarcinogenesis,establishingmorerelevantmousecancermodels,andexaminingnovelapproachestocancertreatmentstoobtainthemaximumvalueforcancerpatients.Weenvisagethatfuturedevelopmentsandapplicationsinmousetransgeniccancermodelingwillbefocusedprimarilyintwoareas.OnesuchareaofcurrentandfutureintenseresearchwillbeconcentratedontheuseoftheCRISPR/Cas9systemasthemostversatileandadaptabletransgenictechnologytodateproducingtransgenicmicethatresembletheexactstepsofhumancarcinogenesis.Thesequencedataforanindividualpatienttumor,whichcannowbeobtainedinamorecost-effectiveway,canbefunctionallyvalidatedusingCRISPR/Cas9transgenicinvitroandinvivomousemodels.Thus,alltheimprovementsandresultsfromthesenovelmousecancermodelswillhopefullyhelptorevealmoregenotype-specificsusceptibilitiesofparticularhumancancertypestofinallyenablemorepersonalized,genotype-basedtreatmentsforcancerpatients. Conversely,sinceincreasinglymoreisknownabouttheimportanceofthetumormicroenvironment,notonlyontumorgrowthbutalsoonthelocalandsystemicresponsetotherapy,thereisamoreextensivedemandforthedevelopmentofmousemodelsthatmoreaccuratelyrepresentthehumantumormicroenvironment.HumanizedmousemodelswithimplantedPDXandhumanmicrobiotawouldbringcancerimmunotherapyresearchonestepfurther,enablingtheexaminationofthecomplexinteractionbetweenthetumor,immunesystem,andmicrobiomeasonesysteminthepatient.Thisapproachcouldpotentiallybeusedtoscreenforeffectiveimmunotherapeuticagentsorcombinations,tostudymechanismsofresistancetoimmunotherapiesandtostudyapproachesonhowtoturnimmunologicallycoldtumorsintohotones.Althoughconceptuallydiverse,bothapplicationshavethefinalaimtotailortherapeuticregimensbasedonspecificmolecularprofilesoftumors.Themajorityoftheapplicationsofthesetwoapproachesarestillatthepreclinicalstage,buttheyshowgreatpromisetosoonbecomemoreclinicallyrelevantastheydeveloptowardamorematurestage. Takentogether,forthcomingimprovementsinmousecancermodelsmightpresentonesuccessfulpathwaytopreciseindividualizedcancertherapy,leadingtoimprovedcancerpatientsurvivalandqualityoflife. AuthorContributions Allauthorswereinvolvedwiththeconceptionanddesignofthemanuscript,manuscriptwriting,andfinalapprovalofthemanuscript. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. 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