Targeted mutagenesisKnockout mice(a)UsingrecombinantDNA(b)Purebredagoutimicearematedtoproducetechniques,a gene specifyinganearlyembryo(ablastocyst).Embryonicstem(ES)cellsfromtheinnercellmassofresistancetothedrugneomycinthe blastocyst are culturedto increasetheir(neo)isinsertedintoanexonofthegeneof interest.number.neoexonneoBlastocystFig.17.820-16
20-16 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Targeted mutagenesis Targeted mutagenesis Knockout mice Knockout mice Fig. 17.8
neoBlastocystESculture(c)CulturedEScellsaretreatedwiththeclonedexondisruptedbyneo.Inasmallnumberofthesecells,homologousrecombinationswapsawild-tvpeexononamousechromosomeinthe EScells with the disruptedversionoftheexon (aknockoutallele),ThecellswithknockoutsareselectedbyexposingtheESculturetoneomycin,andthey aregrownintocolonies.HomologousrecombinationSelectcellswithknockoutalleleAddedDNAconstructwith neomycin,53EScellchromosomewithwild-typealleleEScellchromosomewithknockoutalleleFig.17.820-17
20-17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 17.8
EscellchromosomewithknockoutalleleInjectinto(d)PurebredblackmicearematedtoproduceblastocystablastocystthatisremovedfromthefemaleFostermother(e)About 10 EScells with theknockout allelefromthesamecolonyareinjectedintotheblastocystfromtheblackmice.Thisblastocystisthenplacedintheuterusofanotherblackfemale,whereitcandevelopintoalive-bornmouse.(f)Someofthemicethatdevelopedfromtheinjectedblastocystswillbechimeras,withsomecellsderivedfromtheblackparentsand othercells derivedfromthe agouti Escell culture.Chimericanimals are matedwithpurebredblackmice.lfthegermlineoftheChimerachimeracontainsagouti-derived cells,thensomeoftheoffspringofthismatingwillbeagouti (whichisdominanttoblack).Theseagouti progenyshould be heterozygousfortheknockoutallele;this can be checkedby analyzing DNAfrom thetail.Agouti brothersandsisterswiththeknockoutallelecansubsequentlybematedwitheachothertoproducemicehomozygousfortheknockoutallele(notshown)20-18Fig.17.8
20-18 Fig. 17.8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
Geneknock out cassetteNon-homologousendHomologousjoining(NHEJ)recombination HRPositiveFalse positive20-19
20-19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Homologous recombination (HR) Non-homologous end joining (NHEJ) Positive False positive Gene knock out cassette
2.Hypomorphicmutations（亚效突变）Partial loss-of-function alleleUseful for inferring the function of genes expressed duringdifferent times in developmentwinglessEarlyLaterembryonicdevelopmentdevelopment(wing)20-20
20-20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 2. Hypomorphic Hypomorphic mutations ( mutations (亚效突变) Partial loss Partial loss-of-function allele function allele Useful for inferring the function of genes expressed during Useful for inferring the function of genes expressed during different times in development different times in development wingless Early embryonic development Later development (wing)