Tet-On Advanced Inducible Gene Expression Systems （pTet on advanced，pTRE Tight，pTRE Tight Luc

文档简介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: