Invitrogen GeneSwith 哺乳动物诱导系统pSwitch 说明书

文档简介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