预览加载中，请您耐心等待几秒...

TALENs介导的山羊BLG基因敲除和hLF基因敲入的研究.docx

预览
1/3
2/3
3/3

在线预览结束，喜欢就下载吧，查找使用更方便

下载提示 文本预览

如果您无法下载资料，请参考说明：

1、部分资料下载需要金币，请确保您的账户上有足够的金币

2、已购买过的文档，再次下载不重复扣费

3、资料包下载后请先用软件解压，在使用对应软件打开

TALENs介导的山羊BLG基因敲除和hLF基因敲入的研究 Title:TALEN-mediatedGeneKnockoutandKnock-inofBLGGeneandhLFGeneinGoats Abstract: Transcriptionactivator-likeeffectornucleases(TALENs)haveemergedaspowerfultoolsforgenomeeditingduetotheirprecisionandefficiency.Inthisstudy,weaimedtoinvestigatetheapplicationofTALENsinmodifyingtheβ-lactoglobulin(BLG)geneandintroducingthehumanlactoferrin(hLF)geneingoats.KnockingouttheBLGgeneingoatscouldeliminatetheproductionofallergenicproteins,whileintroducingthehLFgenecouldenhancethenutritionalvalueofgoat'smilk.Inthispaper,wedescribethedesignandgenerationofTALENconstructs,aswellasthesuccessfulknockdownoftheBLGgeneandknock-inofthehLFgeneingoatembryosusingTALENs.Furthermore,wehighlightthepotentialbenefitsandchallengesofusingTALEN-mediatedgenomeeditinginlivestock. Introduction: Goat'smilkisavaluablesourceofnutrients,butitcontainsallergenicproteinssuchasβ-lactoglobulin(BLG),whichcaninduceallergicreactionsinsusceptibleindividuals.KnockingouttheBLGgeneingoatswouldeliminateorreducetheproductionofBLG,resultinginhypoallergenicmilk.Ontheotherhand,humanlactoferrin(hLF)isamultifunctionalproteinwithvarioushealthbenefits.IntroducingthehLFgeneintothegoatgenomewouldenhancethenutritionalvalueofgoat'smilk.TALENsarecustomizablenucleasesthathavebeenwidelyusedforprecisegenomeeditinginvariousorganisms,includinglivestockspecies.Inthisstudy,weaimedtoutilizeTALENstoknockouttheBLGgeneandknockinthehLFgeneingoats. Methods: DesignandconstructionofTALENvectors:TALENsweredesignedtotargetspecificsiteswithintheBLGgeneusingamodularassemblyapproach.TheTALENpairswereclonedintoexpressionvectorsandthenvalidatedfortheiractivityinculturedgoatsomaticcells. Embryomicroinjection:FertilizedgoatembryoswerecollectedandsubjectedtomicroinjectionwithTALENmRNA,followedbyinvitroculturetoallowfordevelopmenttotheblastocyststage.GenomicDNAwasisolatedfromtheresultingblastocystsforgenotypinganalysis. EvaluationofBLGgeneknockout:TheefficiencyofBLGgeneknockoutwasevaluatedbyPCR,followedbyDNAsequencingandWesternblota