Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo 英文参考文献.docVIP

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Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo 英文参考文献.doc

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Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo 英文参考文献

HeritableandLineage-SpecificGeneKnockdownin ZebrafishEmbryo MeiDong1,Yan-FangFu1,Ting-TingDu1,Chang-BinJing1,Chun-TangFu1,YiChen1,YiJin1,MinDeng1*, TingXiLiu1,2,3 * 1LaboratoryofDevelopmentandDiseasesandKeyLaboratoryofStemCellBiologyandStateKeyLaboratoryforMedicalGenomics,InstituteofHealthSciences,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Shanghai Jiao Tong University School of Medicine, and Shanghai Institute of Hematology, RuiJin Hospital,ShanghaiJiaoTongUniversitySchoolofMedicine,Shanghai,People’sRepublicofChina,2ShanghaiStemCellInstitute,ShanghaiJiaoTongUniversitySchoolof Medicine,Shanghai,People’sRepublicofChina,3ModelOrganismDivision,E-InstitutesofShanghaiUniversities,Shanghai,People’sRepublicofChina Abstract Background:Reducedexpressionofdevelopmentallyimportantgenesandtumorsuppressorsduetohaploinsufficiencyor epigeneticsuppressionhasbeenshowntocontributetothepathogenesisofvariousmalignancies.However,methodology thatallowsspatio-temporallyknockdownofgeneexpressioninvariousmodelorganismssuchaszebrafishhasnotbeen wellestablished,whichlargelylimitsthepotentialofzebrafishasavertebratemodelofhumanmalignantdisorders. PrincipalFinding:Here,wereportthatmultiplecopiesofsmallhairpinRNA(shRNA)areexpressedfromasingletranscript thatmimicsthenaturalmicroRNA-30eprecursor(mir-shRNA).Themir-shRNA,whenmicroinjectedintozebrafishembryos, induced an efficient knockdown of two developmentally essential genes chordin and a-catenin in a dose-controllable fashion.Furthermore,wedesignedanovelcassettevectortosimultaneouslyexpressanintronicmir-shRNAandachimeric redfluorescentproteindrivenbylineage-specificpromoter,whichefficientlyreducedtheexpressionofachromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts,respectively. Significance: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue- specific manner or to establish anima