Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome 英文参考文献.docVIP

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Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome 英文参考文献.doc

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Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome 英文参考文献

SimpleMethodsforGeneratingandDetectingLocus- SpecificMutationsInducedwithTALENsintheZebrafish Genome TimothyJ.Dahlem1.,KazuyukiHoshijima1.,MichaelJ.Jurynec1,DerrickGunther1,ColbyG.Starker2, AlexandraS.Locke1,AllisonM.Weis1,DanielF.Voytas2,DavidJonahGrunwald1* 1DepartmentofHumanGenetics,UniversityofUtah,SaltLakeCity,Utah,UnitedStatesofAmerica,2DepartmentofGenetics,CellBiology,andDevelopmentandCenter forGenomeEngineering,UniversityofMinnesota,Minneapolis,Minnesota,UnitedStatesofAmerica Abstract The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simpleandefficientmethodsforinducing,detecting,andrecoveringmutationsatvirtuallyanylocusinthezebrafish.Briefly, double-strandDNAbreaksareinducedatalocusofinterestbysyntheticnucleases,calledTALENs.Subsequenthostrepair oftheDNAlesionsleadstothegenerationofinsertionanddeletionmutationsatthetargetedlocus.Todetecttheinduced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitivemethodfordetectingthepresenceofnewlyarisingsequencepolymorphisms.AstheDNAbindingspecificityofa TALENisdeterminedbyacustomdesignedarrayofDNArecognitionmodules,eachofwhichinteractswithasingletarget nucleotide,TALENswithveryhightargetsequencespecificitiescanbeeasilygenerated.Usingfreelyaccessiblereagents andWeb-basedsoftware,andaverysimplecloningstrategy,aTALENthatuniquelyrecognizesaspecificpre-determined locusinthezebrafishgenomecanbegeneratedwithindays.HerewedevelopandtesttheactivityoffourTALENsdirected atdifferent targetgenes. Usingtheexperimentalapproach described here,everyembryo injected withRNAencoding a TALENwillacquiretargetedmutations.Multipleindependentlyarisingmutationsareproducedineachgrowingembryo, andupto50%ofthehostgenomesmayacquireatargetedmutation.Uponreachingadulthood,approximately90%of th