pRevTet-On 说明书

文档简介四环素调控系统pRevTet-OnpRevTet-On型号载体名称出品公司载体用途VSC0469pRevTet-OnClontech四环素调控系统Description:pRevTet-Onisaretroviralvectorexpressingthereversetetracycline-controlledtransactivator（rtTA）fromtheCMVpromoter.ThisvectorisderivedfrompLNCX,aretroviralvectorcreatedusingelementsofMoloneymurineleukemiavirus（MoMuLV）andMoloneymurinesarcomavirus（MoMuSV）asdescribed（1）.rtTAisafusionofaminoacids1207ofthereversetetrepressor（rTetR）andthenegativelychargedC-terminalactivationdomain（130aminoacids）oftheVP16proteinofHerpesSimplexVirus.rTetRwasderivedfromTetRanddiffersbyfourpointmutations,whichareresponsibleforitsoppositeresponsetodoxycycline.The5'viralLTRcontrolsexpressionofthetranscriptthatcontainsΨ+（theextendedviralpackagingsignal）andtheneomycinresistancegene（Neor）forantibioticselectioninmammaliancells.rtTAisderivedfromvectorsdescribedpreviously（24）.pRevTet-OnalsoincludestheE.coliAmprgeneforantibioticselectioninbacteria.Use:pRevTet-OncanbeusedtoestablishstableTet-Oncelllinesviaretrovirus-mediatedgenetransfer（5）.Retroviralgenetransferallowsthehighlyefficienttransductionofvirtuallyalldividingcelltypes.TheRevTetSystemsarealsosuitableforestablishingtransgenicanimals.IncombinationwiththepRev-TREretroviralexpressionvector,ageneofinterestcanbeinduciblyexpressedathighlevelsinresponsetovaryingconcentrationsofthetetracyclinederivativedoxycycline（Dox）.rtTAbindstotheTet-responseelement（TRE）,thusactivatingtranscriptioninthepresenceofDox.TheresponseofrtTAtoDoxisthusoppositetotheresponseoftTA.AsDoxisremovedfromtheculturemedium,transcriptionfromtheinduciblepromoteristurnedoffinahighlydose-dependentmanner.pRevTet-Onlackstheviralgenesgag,pol,andenv,whicharesuppliedbythepackagingcellline.Itcanbetransfectedintoahightiterpackagingcelllineandtherebymediateproductionofinfectious,replication-incompetentretroviralparticles（1,67）.ThetranscriptproducedbythepRevTet-Onconstructisrecognizedbytheviralstructuralproteinsexpressedinapackagingcelllineandpackagedintoinfectiousretroviralparticles.BecausetheRNAtranscriptpackagedintheseparticlesdoesnotcontaintheviralgenes,itcannotreplicateinthetargetcellsthatitinfects.Thelevelofinductionincellpopulationsinfectedwiththisvectordependsontheefficiencyofinfection,thesiteofintegration,andthetiterofthevirus.Viralsupernatantswithtiters>105cfu/ml首ldbeproducedtoachievehigh-levelinduction.质粒图谱: