A Tightly Controlled Conditional Knockdown System Using the Tol2 Transposon-Mediated Technique 英文参考文献.docVIP

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A Tightly Controlled Conditional Knockdown System Using the Tol2 Transposon-Mediated Technique 英文参考文献.doc

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A Tightly Controlled Conditional Knockdown System Using the Tol2 Transposon-Mediated Technique 英文参考文献

ATightlyControlledConditionalKnockdownSystem UsingtheTol2Transposon-MediatedTechnique TokuichiIguchi1,2,HideshiYagi1,Chen-ChiWang1,MakotoSato1,2,3* 1Division of Cell Biology and Neuroscience, Department of Morphological and Physiological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan, 2ResearchandEducationProgramforLifeScience,UniversityofFukui,Fukui,Japan,3FacultyofMedicalSciences,ChildDevelopmentResearchCenter,Universityof Fukui,Fukui,Japan Abstract Background:Geneknockdownanalysesusingtheinuteroelectroporationmethodhavehelpedrevealfunctionalaspectsof genes of interest in cortical development. However, the application of this method to analyses in later stages of brain developmentorintheadultbrainisstilldifficultbecausetheamountofinjectedplasmidsinacelldecreasesalongwith developmentduetodilutionbycellproliferationandthedegradationoftheplasmids.Furthermore,itisdifficulttoexclude theinfluenceofearlierknockdowneffects. Methodology/Principal Findings: We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-induciblegeneexpressiontechnique,whichallowsustomaintainatransgeneforalongperiodoftimeand inducetheknockdownofthegeneofinterest.Weshowedthatexpressionoftheendogenousamyloidprecursorprotein (APP)wassharplydecreasedbyourinducible,stablyintegratedknockdownsysteminPC12cells.Moreover,weinducedan acuteinsufficiencyofDab1withoursystemandobservedthatradialmigrationwasimpairedinthedevelopingcerebral cortex.Suchinhibitoryeffectsonradialmigrationwerenotobservedwithoutinduction,indicatingthatoursystemtightly controlledtheknockdown,withoutanyexpressionleakageinvivo. Conclusions/Significance:Oursystemenablesustoinvestigatethebrainatanyofthelaterstagesofdevelopmentorinthe adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore,wecanperformknock