利用核糖体基因区打靶载体靶向表达改造型人凝血因子Ⅷ

5018200591972**†(,410078.*.†,E-mail:[e-mail])hF(hF-BDDAK39)pHrneo-BDDAK39,(HT1080),hF-BDDAK39.,pHrneo-BDDAK39hF-BDDAK39HT1080,2.0×10−5,hF-BDDAK39,(32±5)ng·106−1·24h−1.A...,70%.,..,,.,.(pHrneo)(10−4~10−5).,pHrneoA.,A,hF[1,2].[3,4]:()FcDNA,1.2kbmRNA,,F;()F;()F,,vWF.,hFhF[5,6].hFhF-BDDAK39.hF-BDDAK39:()hFcDNAB(∆B760~1639);()5′3′UTR;()5′ApoAI;()hFcDNAATGKozak;()L303EF309S.hF-BDDAK39pHrneo,pHrneo-BDDAK39,pHrneo-BDDAK39(HT1080),hF-BDDAK39.1().TaKaRa,pcD-BDDAK39,PT2,pHrneo,HT-1080ATCC,Roche,α-32P-dCTPPerking-Elmer,ReverseTranscriptionSystemPromega,FCALBIOCHEM,()PIERCE,FELISACEDARLANE,hF.PCRRoche.()pHrneo-BDDAK39.Nhe/PmepcD-BDDAK39,hF-BDDAK39PT2Xba/Pme,pT-BDDAK39;,NhepT-BDDAK39,CMVBGHpoly(A)hF-BDDAK39pHrneoNhe,pHrneo-BDDAK39(1).5018200591pHrneo-BDDAK39pcD-BDDAK39,hF-BDDAK39pCDNA3.1;PT2,XbaPme,PcmvBGHpANhe.pHrneo-:LHA(longhomologousarm),SHA(shorthomologousarm),;EMCV-IRESEMCV;NEO,,NEO.pHrneo-BDDAK39:Nhe/PmepcD-BDDAK39,hF-BDDAK39PT2Xba/Pme,pT-BDDAK39;NhepT-BDDAK39,CMVBGHpoly(A)hF-BDDAK39pHrneoNhe,pHrneo-BDDAK39().BIO-RADGenePulser,AhdDNA20µg,2×106HT1080.:2.0kV,50µF.610cm,10mLDMEM(10%FCS),37,5%CO2.24h10mLDMEM(10%FCS),48h,DMEM(10%FCS),G418(300µg/mL).2d1(10%FCS,G418300µg/mL),14d()PCR./gDNA.gDNA,CMVscreen-rc(5′-AATGGGCGGGGGTCGTTGGGCGGTCA-3′)hrDNAscreen-dn(5′-GGCGATTGATCGGCAAGCGACGCTCAGACAG-3′)(2).:97,5min;97,30s,72,2.5min,10(0.6);97,30s,66,30s,72,2min,25;72,10min.()PCR.0.8%PCR,pGEM-T.primer-T7(5′-TAATACGACTCACTATAGGG-3′)primer-SP6(5′-CATACGATTTAGGTGACACTATAG-3′),ABI377.()Southernblot./PCRgDNA.Southernblot[7],Pvu.pHrneo-BDDAK39,primer-1(5′-CCCGGAAACCTGGCCCTGTCTT-3′)primer-2(5′-TGGGGTACCTTCTGGGCATCCTTC-3′)330bpEMCV-IRES.:97,5min,97,30s,60,20s,72,30s,32;72,10min.,α-32P-dCTP.PCR.()RT-PCRhF-BDDAK39mRNA.TRIZOLRNA.RNAReverseTranscriptionSystem(Promega).,primer-3(5′-GCTTCATCCTACTTTACCAAT-3′)primer-4(5′-TGCAGTGGCCACCCTCAGTAGAG-3′)PCR,450p.95,5min;95,30s,55,30s,72,30s,30,72,10min.()WesternblothF-BDDAK39.,5×,9810min8%SDS-PAGE.PVDF,(hF)().,PBS3,ECL.()hF-BDDAK39.hF-BDDAK39ELISA(CEDARLANELaboratoriesLim-ited).hF-BDDAK39().2:.:.:.screen-rcPcmv,screen-dn,1.5kb.,PvuNEO,7.2kb,EMCV-IRES,1974(3)..3pHrneo-BDDAK39NcopHrneo-BDDAK39,9862,3117,25801083bp4.1~3;MMarker2.2HT1080,HT1080(2).CMVscreen-rcrDNAscreen-dn,1.5kb(2).44PCRgDNA,1.5kb.16,2~5.MDL2000,DNAMarker,1.5kbPCR.2.3Southernblot(2),2Pvu,7230bp,EMCV-IRES,7.2kb.PCRSouthernblot,(5).5Southernblot1HT1080;2~5PCR,7.2kb2.4,2×106,G418260,PCR25,4.(=(×/)/),2.0×10−5.2.5RT-PCRTRIZOLRNA.RNA,oligo(T)RT-PCR.RT-PCR,primer-3/primer-4PCR.hF2(2425),(6),450bp,hF-BDDAK39mRNA.6RT-PCR1HT-1080;2~5;MDL2000,DNAMarker~200kD80kD.,hF-BDDAK3990kD80kD,170kD.hF80kD.,80kD,HT1080(7).7Westernblot1,HT1080;2~5,2.7hF-BDDAK39,hF-BDDAK39(32±5)ng·106−1·24h−1,hF-BDDAK39(143±15)mU·106−1·24h−1.3,10−6~10−8.(embryonicstemcell,ES),ES2[8~10].,2.0×10−5,:()rDNA;().rDNA[11,12][13].,rDNA,;,rDNA.rDNA.,Steele[14],.,Stewart[15]rDNALoxP(RFP)rDNA,.,,rDNA.,[16]:()(positive-negativeselection,PNS),,2~10;()(promoter-trap,PT),1000~5000,PT,,,,.,ESPNS,PT.,,rDNANEOrDNA;.CMVhF-BDDAK39.CMV,,,CMV.,polpol,rDNALEU2,URA3ADE2[17],rDNA.Lopes[11]rDNAGAPDHPGK(phosphoglyceratekinase)SOD(Mn2+-dependentsu-peroxidedismutase)50%.CMV1976(32±5)ng·106−1·24h−1.rDNAhF.,CMVmRNA[18].,CMV[19~21].Jenke[22],CMV.,hF,.,,,A.(:2004CB518800)(:2002BA711A07-08,2002BA711A07-03,2002AA227011)(:31830200).1ChenC,FangXD,ZhuJ,etal.Thegeneexpressionofcoagula-tionfactorinmammaliancelllines.ThrombosisResearch,1999,95:105~115[DOI]2PlantierJL,RodriguezMH,EnjolrasN,etal.Afactormini-genecomprisingthetruncatedintronoffactorhighlyim-provestheinvitroproductionoffactor.ThrombHaemost,2001,86(2):596~6033LynchCM,IsraelDI,KaufmanRJ,etal.Sequencesinthecod-ingregionofclottingfactoractasdominantinhibitorsofRNAaccumulationandproteinproduction.HumGeneTher,1993,4(3):259~2724HoebenRC,FallauxFJ,CramerSJ,etal.Expressionoftheblood-clottingfactor-cDNAisrepressedbyatranscriptionalsilencerlocatedinitscodingregion.Blood,1995,85(9):2447~24545KaufmanRJ.Advancestowardgenetherapyforhemophiliaatthemillennium.HumGeneTher,1999,10(13):2091~2107[DOI]6KaufmanRJ,PipeSW.RegulationoffactorexpressionandactivitybyvonWillebrandfactor.ThrombHaemost,1999,82(2):201~2087SambrookJ,FritschEF,ManiatisT.MolecularCloning:ALabo-ratoryManual.2nded.NewYork:ColdSpringHarborLaboratoryPress,19898BrownJP,WeiW,SedivyJM.Bypassofsenescenceafterdisrup-tionofp21CIP1/WAF1geneinnormaldiploidhumanfibroblasts.Science,1997,277(5327):831~834[DOI]9ArbonesML,AustinHA,CaponDJ,etal.Genetargetinginnormalsomaticcells:Inactivationoftheinterferon-gammarecep-torinmyoblasts.NatGenet,1994,6(1):90~97[DOI]10HansonKD,SedivyJM.Analysisofbiologicalselectionsforhighefficiencygenetargeting.MolCellBiol,1995,15(1):45~5111LopesTS,KlootwijkJ,VeenstraAE,etal.High-copy-numberintegrationintotheribosomalDNAofSaccharomycescerevisiae:Anewvectorforhigh-levelexpression.