RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration 英文参考文献.docVIP

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RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration 英文参考文献.doc

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RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration 英文参考文献

RNAInterferenceIsResponsibleforReductionof TransgeneExpressionafterSleepingBeautyTransposase MediatedSomaticIntegration ChristinaRauschhuber1,AnjaEhrhardt1,2* 1MaxvonPettenkofer-Institute,DepartmentofVirology,Ludwig-Maximilians-UniversityMunich,Munich,Germany,2InstituteofVirologyandMicrobiology,Centerfor BiomedicalEducationandResearch,DepartmentofHumanMedicine,FacultyofHealth,UniversityWitten/Herdecke,Witten,Germany Abstract Background: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammaliancellsinfunctionalgenomicsandtherapeuticgenetransfer.FortheSleepingBeauty(SB)transposasesystemit was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occursaftersomaticintegration.Thiscouldleadtoformationofdouble-strandedRNAspotentiallypresentingtargetsfor theRNAinterference(RNAi)machineryandsubsequentlyresultingintosilencingofthetransgene.Therefore,weaimedat investigatingtransgeneexpressionupontranspositionunderRNAinterferenceknockdownconditions. PrincipalFindings:ToestablishRNAiknockdowncelllineswetookadvantageoftheP19protein,whichisderivedfromthe tomatobushystuntvirus.P19bindsandinhibits21nucleotideslong,small-interferingRNAsandwasshowntosufficiently suppressRNAi.WefoundthattransgeneexpressionuponSBmediatedtranspositionwasenhanced,resultingintoa3.2-fold increasedamountofcolonyformingunits(CFU)aftertransposition.Incontrast,ifthetransgenecassetteisinsulatedfrom theinfluenceofchromosomalpositioneffectsbythechicken-derivedcHS4insulatingsequencesorwhenapplyingtheForg Princetransposonsystem,thatdisplaysonlynegligibletranscriptionalactivity,similarnumbersofCFUswereobtained. Conclusion:Insummary,weprovideevidenceforthefirsttimethataftersomaticintegrationtransposonderivedtransgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the moleculardesignoftheSBtransposasevectorsystem. Citation:Rauschhube