pRevTet-off说明书

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pRevTet-off型号载体名称出品公司载体用途VSC0470pRevTet-offClontech四环素调控系统载体描述:pRevTet-Offisaretroviralvectorexpressingthetetracycline-controlledtransactivator（tTA）fromtheCMVpromoter.ThisvectorisderivedfrompLNCX,aretroviralvectorcreatedusingelementsofMoloneymurineleukemiavirus（MoMuLV）andMoloneymurinesarcomavirus（MoMuSV）asdescribed.tTAisafusionofaminoacids1207ofthetetrepressor（TetR）andthenegativelychargedC-terminalactivationdomain（130aminoacids）oftheVP16proteinofHerpesSimplexVirus.The5'viralLTRcontrolsexpressionofthetranscriptthatcontainsY+（theextendedviralpackagingsignal）,andtheneomycinresistancegene（Neor）forantibioticselectioninmammaliancells.tTAisderivedfromvectorsdescribedpreviously.pRevTet-OffalsoincludestheE.coliAmprgeneforantibioticselectioninbacteria.pRevTet-OffcanbeusedtoestablishstableTet-Offcelllinesviaretrovirus-mediatedgenetransfer.Retroviralgenetransferallowsthehighlyefficienttransductionofvirtuallyalldividingcelltypes.TheRevTetSystemsarealsosuitableforestablishingtransgenicanimals.IncombinationwiththepRev-TREretroviralexpressionvector,ageneofinterestcanbeinduciblyexpressedathighlevelsinresponsetovaryingconcentrationsoftetracycline（Tc）orTcderivativessuchasdoxycycline（Dox）.tTAbindstotheTet-responseelement（TRE）,thusactivatingtranscriptionintheabsenceofTcorDox.AsTcorDoxisaddedtotheculturemedium,transcriptionfromtheinduciblepromoteristurnedoffinahighlydose-dependentmanner.pRevTet-Offlackstheviralgenesgag,pol,andenv,whicharesuppliedbythepackagingcellline.Itcanbetransfectedintoahightiterpackagingcelllineandtherebymediateproductionofinfectious,replication-incompetentretroviralparticles.ThetranscriptproducedbythepRevTet-Offconstructisrecognizedbytheviralstructuralproteinsexpressedinapackagingcelllineandpackagedintoinfectiousretroviralparticles.BecausetheRNAtranscriptpackagedintheseparticlesdoesnotcontaintheviralgenes,itcannotreplicateinthetargetcellsthatitinfects.Thelevelofinductionincellpopulationsinfectedwiththisvectordependsontheefficiencyofinfection,thesiteofintegration,andthetiterofthevirus.Viralsupernatantswithtiters>105cfu/ml首ldbeproducedtoachievehigh-levelinduction.质粒图谱:
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