BD RevTet System使用说明
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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:
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