wnt16基因crisprcas9敲除对斑马鱼骨骼发育影响的初步研究

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1、ABSTRACTOsteoporosisisadiseasecharacterizedbylowbonemassandmicroarchitecturaldeteriorationofbonetissueleadingtoincreasedriskoffracture．Osteoporosiswasdefmedclinicallythroughthemeasurementofbonemineraldensity(BMD)，whichremainthesinglebestpredictoroffracture．Inlastdecade，alargenumberofge

2、nome—wideassociationanalysis(GWASs)andmeta—analysisidentifiedseveralsinglenucleotidepolymorphisms(SNPs)mutationlocatedinthewntl6gene(7q31)significantlyassociatedwithBMD，anditwasconfirmedinwntl6knockoutmicemodel．However,themolecularmechanismofwntl6regulatedskeletaldevelopmentinvivoisunc

3、lear,especiallyintheearlyperiodofembryonicdevelopment．InOurresearchCRISPRgeneeditingtechnologywasusedtoknockoutwntl6geneinzebrafish，andthentheeffectsof耽fJG／-geneonskeletaldevelopmentwascheckedtofurtherrevealthemolecularmechanismofbonedevelopment．Inordertoimprovetheefficiencyoftarget，tw

4、otargetgRNAswereinjectedintozebrafishembryoatO一1celllevel，andthentargetactivitywasconfirmedwithT7E1enzymeandDNAsequencingafter50hpf(hourspost—fertilization)．Therewere4mutantsofwntl6genetobesuccessfullyidentifiedinF0adultzebrafishbyTA—clonesequencing．TheF0mutantszebrafishwerehybridizedw

5、ithwildtypetoobtainF1generation，then5differentmutationswerescreenedintheF1generationadultzebrafish．NoframeshiftmutationswerefoundinWntl6(21)F1andwntl6(7)F1，butapartofaminoacidsequencechanged．ItsproteinspatialstructurewaspredictbyPhyreandfoundanalphahelixstructurechanged．Thesamegenotype

6、ofmalesandfemaleszebrafishwerehybridizedtoobtainF2generationandthefemaleshomozygousmutantsTIT万方数据wereidentifiedinwntl6(21)F2．CartilagewasstainedbyAlcianblueinWntl6(21)F2andwntl6(14)F2adultzebrafishat10dpfandalongerMeckel’Scartilageandabendingspinewasfoundinwntl6(21)F2comparedwithwildty

7、pe．Theseresultssuggestthatwntl6affectbonedevelopmentofzebrafish．Keywords：wntl6，CRISPR／CAS9，skeleton，zebrafishIV万方数据目录中文摘要IABSTRACTIII。．前言11．日Ⅱ舌1．1wntl6基因调节骨代谢的研究进展11．1．1骨质疏松症简介一11．1．2WNTl6基因与人类骨密度关联．21．1．3wntl6KO小鼠骨密度下降．21．1．4wntl6敲降斑马鱼造血干细胞分化缺陷31．1．5wntl6基因调节骨代谢机制分析61．2CRISPR／CAS9基因