BD RevTet System使用说明

文档简介BDRevTetSystem使用说明pRevTet-On，pRevTet-Off，pRevTet-Off-IN，pRevTREIntroductionTheBDRevTet-OnTM&BDRevTet-OffTMSystemscombinetheadvantagesofretrovirus-mediatedgenetransferwiththeproventetracycline-regulatedcontrolofBDTet-OnTMandBDTet-OffTMGeneExpressionSystems.BDRevTetTMallowsthefastandefficientestablishmentofregulatedgeneexpressionsystemsinawidevarietyofcelltypes.EachRevTetSystemisacompleteretroviralgeneexpressionsystemcontainingaretroviralregulator（r）tTAvector,aretroviralresponsevector,acontrolvector,andapackagingcellline.LikeourBDRetro-XTMSystem（#631508）,theRevTetSystemscontaintheBDRetroPackTMPT67PackagingCellLine（#631510）forthesafe,efficientproductionofinfectious,replication-incompetentretrovirus.TheRevTetSystemsprovideapowerfulandconvenientapproachtosettingupthetetracycline（Tc）-inducible,high-levelgeneexpressionsystemsfirstdescribedbyGossenandBujard（1992）.Retroviral-mediatedtet-regulationTheTetSystemsarebasedontwoelementsfromthetetoperonoftheE.coliTn10transposontheTetrepressorprotein（TetR）andthetetoperatorDNAsequence（tetO）.ThegenetobeexpressedisclonedintothepRevTREresponsevectordownstreamofthetetracycline-responsiveelement（TRE）,whichcontainssevendirectrepeatsofthe42-bptetOsequenceandtheminimalimmediateearlypromoterofcytomegalovirus（PminCMV）.Thetwosystemsdifferintheirregulatoryvector（Figure1）.TheRevTet-OffSystemusesthepRevTet-OffVector,whichcontainsthetTAregulatoryelement,afusionofTetRandtheVP16activationdomain.TheBDRevTet-OnTMSystemusespRevTet-On,basedonthereversetTA（rtTA）.BothsystemsusethepRevTREresponsevector.AlltheRevTetVectorsarederivedfrompLNCX,aBDRetro-XTMvectorcapableofproducinghigh-titervirusintheappropriatepackagingcelllines.IntheRevTet-OffSystem,tTAbindstheTREandactivatestranscriptionintheabsenceofTcortheTcderivativedoxycycline（Dox）.Thus,asTcorDoxisaddedtotheculturemedium,transcriptionisturnedoffinadose-dependentmanner（Figure1）.Conversely,theRevTet-OnSystemallowsgeneexpressiontobeactivatedbytheadditionofDox.Thesetwocomplementarysystemsallowyoutochoosetheonethatbestsuitsyourneeds.BenefitsandapplicationsofTc-mediatedinductionInmostinduciblemammaliangeneexpressionsystems（e.g.,inductionbyheavymetals,steroidhormones,orheatshock）,inductionisnonspecificandexpressionlevelscannotbepreciselyregulated.Inaddition,thesesystemsaregenerallyleakyinthe“off”state,andtheinducingagentitselfmaybecytotoxicorhavepleiotropiceffectsonresults.Incontrast,regulationofgeneexpressionbytheheterologousbacterialcontrolelementsintheRevTetSystemsisveryspecific.Furthermore,thelevelsofTcorDoxrequiredforthefullrangeofgeneexpressionarenotcytotoxicandhavenosignificanteffectoncellproliferationoranimalgrowth,evenwithcontinuoustreatment（Bohletal.,1997;Mayfordetal.,1996）.TheuseofseparateregulatoryandresponsevectorsintheRevTetSystems质粒图谱: