Transgenic Mice - Learn Genetics (Utah)

During the 1980s, Capecchi devised a way to change or remove any single gene in the mouse genome, creating strains of mice that pass the altered gene from ... DonateHomeGeneticScienceandSocietyTransgenicMiceTransgenicMiceMarioR.Capecchi,Ph.D.,oftheUniversityofUtah, wonthe2007NobelPrizeinPhysiologyorMedicine.CapecchisharedtheprizewithOliver SmithiesofUniversityofNorthCarolina,ChapelHill,andSirMartinEvansofCardiffUniversityintheUK.TheprizerecognizedCapecchi'spioneeringworkon"knockoutmouse"technology,agene-targetingtechniquethathasrevolutionizedgeneticandbiomedicalresearch, allowingscientiststocreateanimalmodelsforhundredsofhumandiseases.WatchavideoofTheUniversityofUtahNobelPressConference.What'sTheMotivation?Asachild,CapecchiwanderedhomelessinItaly.Asaresearcher,hisfirstattemptsatgenetargetingweredeemednotreadyforfunding bytheNationalInstitutesofHealth.Capecchiisanindividualwhosepersonallifeprovesthatwhilesomeeventsarenotprobable, anythingispossible.ReadMario'sstory.Duringthe1980s,Capecchidevisedawaytochangeorremoveanysinglegeneinthemousegenome,creatingstrainsofmice thatpassthealteredgenefromparenttooffspring.Intheyearssince,these"transgenic"and"knockout"micehavebecome commonplaceinthelaboratory.Capecchi'spioneeringworkingenetargetinghastaughtusmuchabouthowthebodybuilds—andrebuilds—itself.Hehasgiven scientistsworldwidethetoolstomakeimportantdiscoveriesabouthumandiseases,fromcancertoobesity.Andhehasraisedakeyquestionforthefutureofhumanmedicine:ifwecanreplaceaperfectlygoodgenewithamutatedone, canwealsogotheotherway,replacingproblemgeneswiththosethatwork? Mario'sLabatWorkWhatmakesanarmanarm?Capecchi'sresearchteamisworkingonansweringthatquestionusinggenetargeting.Theyhave systematically"knockedout"asetofgenesinmice,calledhomeoticgenes,whichgovernbodypatterningduringdevelopment. Forexample,oneofthelab'smostrecentgeneticdiscoveriesmayexplain whywelackspareribs.Findoutmoreabouthow homeoticgenesworkinHomeoticGenesandBodyPatterns.HowtoBuilda'Knockout'MouseYOURGOAL:Youarestudyinghowaparticulargene,namedOhNo,mightplayaroleinpanicattacks.Youwanttostudymicethataremissingthis gene.To"knockout"theOhNogene,youwillreplaceitwithamutatedcopythatdoesn'twork. Here'show:1.IsolateStemCells IsolateembryonicstemcellsthatoriginatedfrommalebrownmicewithanormalOhNogene(blue).2.AddInactiveGeneWithMarkerTothesecells,addacopycontainingamutated,inactiveOhNogene(red),andadrugresistancemarkergene(pink).3.SimilarGenesNaturallySwapBymechanismsthatarenotcompletelyunderstoodyet,similargeneswillswapplaces.The OhNogeneplusdrugresistancemarkergeneisincorporatedintothegenome,andthenormalversion iskickedout.Thisprocessiscalledhomologousrecombination.4.AddDrugCellsthathaven'tincorporatedtheinactiveOhNogenedon'thavethedrugresistancemarkergene(pink).Addingthedrugkillscellswithoutthemarker,leavingyouwithonlycellsthathaveaninactiveversionoftheOhNogene.5.GrowChimericMiceBytransplantingstemcellsthatcarrytheinactiveohNogeneintoawhitemouseembyro,you'llcreatewhatiscalledachimera. Chimerashavepatchesofcellsthroughouttheirbodiesthatgrewfromwhitemousecellsandpatchesthatgrewfrombrownstemcells. SomeofthecellsthathavetheinactiveOhNogenemaydevelopintoreproductivecells.Chimerasareeasytoidentifybecausetheyhavebothbrownandwhitepatchesoffur.6.MateMaleChimeraIfamalechimerahassomereproductivecells(sperm)thatoriginatedfromthebrownstemcells,hewillproduce somebrownoffspringwhenmatedwithawhitefemale.7.TestandBreedBrownOffspringHalfofthebrownoffspringwillhaveacopyoftheinactiveOhNogeneinalloftheircells—including theirreproductivecells.ThesemicehaveonenormalcopyoftheOhNogenefromtheirmother(notshown)andoneinactive copyfromtheirfather.Sohalfoftheirreproductivecellswillcontainanormalcopy,andhalfwillcontainaninactivecopy.ThesemicecanbeidentifiedbyperformingDNAsequencingintheirOhNogenesandthenbredwitheachother.8.You'veMadeaKnockoutMouse Onefourthofyourresultingoffspingwillhavetwocopiesofthe"knocked-out"orinactiveOhNogene.Youcannow studythesemicetodeterminehowlackingtheOhNogenemayaffectpanicattacks.FundingThisworkwassupportedbyScienceEducationPartnershipAwards(Nos.R25RR023288and1R25GM021903)fromtheNationalInstituteofGeneralMedicalSciencesoftheNationalInstitutesofHealth.ThecontentsprovidedherearesolelytheresponsibilityoftheauthorsanddonotnecessarilyrepresenttheofficialviewsofNIH.APAformat:GeneticScienceLearningCenter.(2013,July15)TransgenicMice. RetrievedApril01,2022,fromhttps://learn.genetics.utah.edu/content/science/transgenicCSEformat:TransgenicMice[Internet].SaltLakeCity(UT):GeneticScienceLearningCenter;2013 [cited2022Apr1]Availablefromhttps://learn.genetics.utah.edu/content/science/transgenicChicagoformat:GeneticScienceLearningCenter."TransgenicMice."Learn.Genetics. July15,2013.AccessedApril1,2022.https://learn.genetics.utah.edu/content/science/transgenic.