annual plant reviews gene targeting

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1、c04BLBK148-KnightFebruary17,200921:12CharCount=AnnualPlantReviews(2009)36,76112www.interscience.wiley.comdoi:10.1111/b.9781405181891.2009.00004.xChapter4GENETARGETINGYasukoKamisugiandAndrewC.CumingCentreforPlantSciences,UniversityofLeeds,LeedsLS29JT,UKAbstract:Physcomitrellapatensisamo

2、delplantforcomparativegenomicanalysis,andistheonlyplantmodelinwhichgenetargetingthereplacementofanendogenousgenebyageneticallyalteredvarianttransgeneoccurswithhigheffi-ciency.Wedescribetheparametersthatdeterminetheefficiencyofgenetargetinganddiscussthenatureoftheintegrationeventsthatoccu

3、rfollowingtransforma-tionofP.patens.IntegrationoftransgenesutilisesendogenousmechanismsfortherepairofDNAdouble-strandbreaks,andthepreferentialintegrationoftrans-formingDNAathomologoussitesbyP.patensindicatesthatthepredominatingmechanismforDNArepairisbasedonhomologousrecombination,rathe

4、rthannon-homologousendjoining,asisthecaseinfloweringplants.P.patensthuspro-videsausefulexperimentalmodelforthestudyoftheHR-mediatedDNArepairpathwayinplants,andunderstandinghowthismechanismoperatesinP.patensmaypavethewayforthedevelopmentofmoreefficientgenetargetingstrategiesforthegeneticm

5、odificationofcropspecies.Keywords:allelereplacement;DNAdouble-strandbreakrepair;genetargeting;homologousrecombination;non-homologousendjoining;Physcomitrellapatens4.1IntroductionGenetargetingdescribesthedeliveryofexogenousDNAtoanorganismsuchthatthisDNAintegrates,asatransgene,ataprecisel

6、ydefinedsiteintherecipientgenome.ThekeyfeatureofsuchgenetictransformationisthatthesiteofDNAintegrationispredetermined.ThisdistinguishestheprocedurefromexperimentalapproachesinwhichpopulationsoftransgenicorganismsaregeneratedthroughthedeliveryofDNAvectorsthatintegrateessentiallyatrandom,

7、butwhosesiteofintegrationinthegenomemaysub-sequentlybedeterminedthroughDNAsequenceanalysisofthetransgeniclocus.GenetargetingrequiresthattheexogenouslysuppliedDNAbeabletoseekoutspecificsequencesinthetargetgenomewithwhichitsharessequencehomology,andthatitistheninsertedintothehomologousg