pCDH-CMV-MCS-EF1-copGFP-T2A-Puro

载体名称:出品公司：pCDH-CMV-MCS-EF1-copGFP-T2A-PuroSBI质粒类型:慢病毒表达载体；cDNA表达载体；双启动子载体克隆方法:多克隆位点，限制性内切酶启动子:CMV载体大小:--5'测序引物及序列:CMV-F:CGCAAATGGGCGGTAGGCGTG3'测序引物及序列:--载体标签:无载体抗性:氨苄青霉素（Ampicillin）筛选标记:GFP、Puromycin克隆菌株:E.colicells(RecA-)推荐:Stbl2,OmniMAX2T1R宿主细胞（系）:常用细胞系，如HeLa,HEK293,HT1080,H1299备注:pCDH-CMV-MCS-EF1-copGFP-T2A-Puro慢病毒表达载体是基于HIV的慢病毒载体；用于cDNA表达和克隆；高效转染细胞，建立稳定细胞系；CMV启动子驱动目的基因的高水平表达，EF1a启动子驱动报告基因的中等水平的表达。产品目录号:CD513B-1稳定性:稳表达组成型/诱导型:组成型病毒/非病毒:慢病毒（HIV)载体质粒图谱和多克隆位点信息pCDH慢病毒载体的包装载体及细胞系:Theexpressionlentivectorcontainsthegeneticelementsresponsibleforpackaging,transduction,stableintegrationoftheviralexpressionconstructintogenomicDNA,andexpressionofthetargetgenesequence.ThepackagingvectorprovidesalltheproteinsessentialfortranscriptionandpackagingofanRNAcopyoftheexpressionconstructintorecombinantviralparticles.Toproduceahightiterofviralparticles,expressionandpackagingvectorsaretransientlyco-transfectedintoproducermammaliancells(e.g.,HEK293cells).ForadetaileddescriptionofSBI’sLentivectorexpressionsystem,pleaserefertotheLentivectorExpressionSystemusermanual.启动子的选择：SBIprovidesacollectionofcDNAcloningandexpressionvectorsforvariousapplications.AgeneofinterestcanbeclonedunderaCMVorEF1promoterwithorwithoutanotherexpressioncassetteforareportergene(copGFPorPuroR).Genescanbeeitherexpressedtransientlythroughtransfectionorstablyexpressedinatargetcelllinethroughtransductionwithpackagedviralparticles.ThemajorconcernofcDNAexpressioninlentivectorsistheefficiencylevelandstabilityofexpressionintargetcelllines.TheCytomegalovirus(CMV)promoterisastrongandmostcommonlyusedviralpromoterthatconstitutivelyexpressesdownstreamgenes.WhiletheCMVpromoterworksperfectlyinthemostcommoncelllines,itshowspoorexpressioninsomestemcelllinesandhematopoieticcelllines(R.F.Doll,1996;E.D.Papadakis,2004).Thehousekeepingelongationfactor1α(EF1)promoterhasbeenshowntoexceedandoutlastCMV-mediatedexpressioninretroviral,lentiviral,andadenoviralvectors,inhematopoieticcelllines(K.Tokushige1997;H.Nakai,1998;C.Teschendorf,2002).EF1alsoperformswellinmostcommoncelllines.MSCVpromoteristhe5’-LTRpromoterofmurinestemcellvirus.WhenaportionoftheU3regionofthe3’HIVLTRwasreplacedwiththeU3regionofMSCVLTR,theresultedhybridHIV/MSCVLTRhasdramaticallyincreasedthetransgeneexpressionlevelinhumanCD34+hematopoieticcells(J.K.Choi,2001).AfterintegrationintogenomicDNA,thispromotertranscribesalongtranscriptwithanintroninthe5’UTRflankedwithsplicedonorandacceptorsitesderivedfromthelentiviralvector.FurtherstudiesfoundthatadditionalCpGmutationsintheMSCVLTRreducedtranscriptionalsilencinginembryonicstemcells(C.S.Swindle,2004).WeconstructedcDNAexpressionvectorswiththeCpG-deficientMSCVincorporatedintothe3’HIVLTR.AfterintegrationintogenomicDNA,3’MSCV/LTRwillreplacethe5’LTRandprovideahighlevelofexpressionofthetargetgeneandreportergenedownstream.