Tet-On Advanced Inducible Gene Expression Systems (pTet on advanced,pTRE Tight,pTRE Tight Luc
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Tet-OnAdvancedInducibleGeneExpressionSystems(pTetonadvanced,pTRETight,pTRETightLuciferase)使用说明书Vectors:pTetonadvanced,pTRETight,pTRETightLuciferaseA.SummaryTheTet-OnAdvancedInducibleGeneExpressionSystemisatightlyregulatedandhighlyresponsivesystemthatproduceson-demand,robustexpressionofyourgeneofinterest(GOI)intargetcells.Thesystemisestablishedintargetcellsbysequentiallytransfectingthemwiththeprovidedvectorsandselectingstablecelllines.TargetcellsthatexpresstheTet-OnAdvancedtransactivator,andthatalsocontainanintegratedTRE-basedexpressionvector(e.g.,pTRE-Tight)willexpresshighlevelsofyourGOIwhenculturedinthepresenceofthesystem’sinducer,doxycycline(Dox)(Figure1).B.ElementsofTet-OnAdvancedInductionBasedontheoriginaltetracycline(Tc)-regulatedtranscriptionaltransactivatorsdescribedbyGossen&Bujard(1992)andGossenetal.(1995),Tet-OnAdvancedisamodifiedtransactivatorproteinthatisoptimizedforexpressioninmammaliancells,andwhichdemonstrateshighersensitivityandfidelitythanpreviousversions(Ur领er,etal.2000).Theinduciblepromoter,PTight,providesforverylowbasalexpressionandtightlycontrolledinduction.TheTet-OnAdvancedtransactivator.ThepTet-OnAdvancedvectorconstitutivelyexpressesthetetracyclinecontrolledtranscriptionaltransactivator,Tet-OnAdvanced(Ur领eretal.,2000).ThisengineeredproteinconsistsofamutantE.coliTetRprotein(rTetR)fusedtothreeminimal"F”-typeactivationdomainsderivedfromtheherpessimplexvirusVP16protein(Baronetal.,1997,Triezenbergetal.,1988).InthepresenceofDox,Tet-OnAdvancedbindstothetetOsequencesinPTight,andactivateshighleveltranscriptionfromthisinduciblepromoter.TheTet-OnAdvancedcodingsequenceisfullysyntheticandutilizeshumancodonpreferencestoincreaseitsexpressionlevelandstabilityinmammaliancells.ThePTightinduciblepromoter.ThisisaninduciblepromoterthatcontrolstranscriptionofyourGOI.ThePTightcompositepromoterwasoriginallydevelopedasthePtet-14promoterinthelaboratoryofDr.H.BujardandconsistsofamodifiedTet-ResponsiveElement(TREmod)containing7directrepeatsofthetetoperatorsequence,tetO,whichisjoinedtoaminimalCMVpromoter(PminCMV).PTightlacksbindingsitesforendogenousmammaliantranscriptionfactors,soitisvirtuallysilentintheabsenceofinduction.InthepresenceofDox,Tet-OnAdvancedbindstightlyandspecificallytoPTightandactivatestranscriptionofthedownstreamGOI(Figure1).质粒图谱:
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