Invitrogen GeneSwith 哺乳动物诱导系统pSwitch 说明书
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InvitrogenGeneSwith哺乳动物诱导系统InvitrogenVectors:pSwitch产品参数:MammalianSelection:Hygromycin载体抗性:氨苄青霉素(Ampicillin)载体描述:Switch-OnExpressionfromtheLowestBasalLevelsTheGeneSwitch?Systemforinduciblemammalianexpressionisidealforexperimentsthatrequiretheabsolutelowestuninducedexpressionlevels.TheexpressionvectorpGene/V5-Hisprovidesaminimalpromoter,GAL4-E1b,consistingofthebindingsitesfortheyeastGal4DNAbindingproteinfollowedbytheTATAsequencefromtheAdenovirusE1bpromoter.Withoutadditionalfactors,theGAL4-E1bpromoteristranscriptionallysilent.ToactivatetranscriptionfromtheGAL4-E1bpromoter,theGeneSwitch?regulatoryproteinisexpressedfromaminimalTKpromoteronthepSwitchvector.TheGeneSwitch?proteinhasthreefunctionaldomains:Gal4DNABindingDomain(Gal4-DBD)tobindtheregulatoryproteintotheGAL4-E1bpromoterTruncatedhumanProgesteroneReceptorLigandBindingDomain(hPR-LBD)thatundergoesaconformationalchangewhenitbindstheprogesteroneantagonist,mifepristoneTranscriptionactivationdomainfromtheNF?Btranscriptionfactorp65(p6D)toactivatetranscriptionfromthesilentGAL4-E1bpromoterIntheabsenceofmifepristone,theconformationofthehPR-LBDregionpreventstheGeneSwitch?regulatoryproteinfromactivatingtranscriptionfromtheGAL4-E1bpromoter.WhenmifepristoneisaddedandbindsthehPR-LBDregion,theGeneSwitch?regulatoryproteinassumesaconformationthatpermitsittostimulatetranscriptionfromtheGAL4-E1bpromoter.InductionoftheGeneSwitch?SystemleadstoactivationofthegeneofinterestonthevectorpGene/V5-His.Inaddition,fourGal4bindingsitesupstreamoftheminimalHSVTKpromoteronpSwitchcanbindthepSwitchregulatoryprotein.Therefore,addingmifepristoneup-regulatesproductionoftheregulatoryprotein.TheincreasedlevelsoftheGeneSwitch?regulatoryproteinresultininductionofthegeneofinterestfrompGene/V5-Histolevelsthatcanapproachthoseofviralpromoters.TheGeneSwitch?Kitoffersexceptionallylowuninducedandhighinducedexpressionlevelsinmammaliancells.ThesystemprovidestheexpressionvectorpGene/V5-Hiswiththeminimal,syntheticGAL4-E1bpromoterthatistranscriptionallysilentuntilactivated.TheGeneSwitch?regulatoryproteinbindstheGAL4-E1bpromotertoactivatetranscriptionupontheadditionofmifepristone.Inaddition,theGeneSwitch?proteinupregulatesitsownexpression,leadingtoamplifiedexpressionofthegeneofinterestfrompGene/V5-His.pGene/V5-Hisoffersseveralfeaturesthatfacilitateexpressionanalysisandpurificationofrecombinantproteinsinmammaliancells:C-terminalV5epitopetagfordetectionwithAnti-VntibodiesC-terminalpolyhistidine(6xHis)tagforrapidpurificationwithnickel-chelatingresinanddetectionwithAnti-His(C-term)AntibodiesTheZeocin?resistancegeneforeffectiveselectioninbothmammaliancellsandE.coli
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