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:2001-11-12;:2001-12-20:(30070837),(30025028)(102-08-08-02)[NationalNaturalSc-ienceFoundationofChina(30070837),NationalScienceFundforDistinguishedYoungScholars(30025028)andtheNationalHigh-TechResearchandDevelop-mentProgram(102-08-08-02)]¹Tel:010-66948882,E-mail:Yangx@nic.bmi.ac.cnCre郝振明,杨晓¹,程萱,周江,黄翠芬(,100071):ÒA1Crepcol2A1-Cre323,1452,PCR10Cre,1912%CreLoxP,CrePCR:col2A1-CreCreLoxPSouthern:Cre;;;:Q373:A:0379-4172(2002)05-0424-06GenerationandCharacterizationofChondrocyteSpecificCreTransgenicMiceHAOZhen-Ming,YANGXiao¹,CHENGXuan,ZHOUJiang,HUANGCu-iFen(GeneticLaboratoryofDevelopmentandDiseases,InstituteofBiotechnologyAcademyofMilitaryMedicalSciences,Beijing100071,China)Abstract:Achondrocytespecifictransgenicconstruct(pcol2A1-Cre)containingthecartilagespecifictypeÒcollagenA1promoter,theCrerecombinasegeneandthepolyAofhumangrowthfactorgenewasgenerated.The913kbDNAfragmentswererecoveredfromNotÑdigestedfragmentsandmicroinjectedinto323fertilizedeggs.Theinjectedeggswereimplantedintotheoviductof14femalemice.Inthe52offsprings,therewere10micecarringthetransgeneidentifiedbyPCR,andtheefficiencywas1912%.Thecol2A1-CretransgenicmicewerecrossedwithaconditionalgenetargetingmicetochecktheCremediatedrecombinationinmultipletis-sues.TheresultsofthePCRanalysissuggestedthattheCrerecombinasewasexpressedonlyincartilaginoustissueandcouldmediatetherecombinationbetweentheLoxPsitesinvivo.TheresultwasfurtherconfirmedbySouthern-blot.Keywords:Cre;transgenicmouse;chondrocyte;tissuespecific(geneknockout),Cre-LoxPCre,38kDa,34bpLoxP,LoxP[1]CreLoxP,LoxPCre,Cre/LoxPCre,,29(5):424~429,2002ActaGeneticaSinicaCre,,[2]SMADsB(TGF-B)Smad3,,SMAD3TGF-B[3,4]SmadsSmadsTGF-B,Cre,CreLoxPDNACre1111ÒA1pcol2A1[5]AndersonBenoitdeCrombruggheCrepIC-Cre[6]HuaGuployADNApBs-hGH[7]VanderbiltM.A.MagnusonDH5A112pcol2A1-Cre[8]CreployApRCHNotÑHindÓpcol2A1610kbÒA1,KpnÑSalÑpRCHDNA,DNA,DNA,/0[9]113[10]11410d14d015cm115ml,400Ll(015%SDS,011mol/LNaCl,0105mmol/LEDTA,0101mmol/LTris#Cl,pH810,K,100Lg/ml),50e,/0[8]DNAPCRCreP1/P2()PCR,:P1:TTGCCTGCATTACCGGTCGATGC;P2:TTGCACGTTCACCGGCATCAACGPCR:94e,4min94e,35s,68e,35s,72e,1min,30cycles72e,10min4eCre368bp115Cre11511PCRCreCreLoxP(Floxed/Floxed,,),P1/P2PCRCrePCR114FloxedLoxP(1),LoxPP3(5cCCTT-1CreFig.1PrimersfordetectingtheCremediatedrecombinationAGTTGAAGCTTATAACTTCG3c)P4(5cGACC-CAAACGTCACCTTCAC3c)DNA,P3/P4PCR,CreCre,LoxP,P3P4234bpDNAFloxedLoxP214kb,PCR,P3P4PCR:94e,4min94e,35s;60e,35s;72e,1min;3072e,10minPCRTALoxPDNA5:Cre42511512Southern,HpaÑEcoRÕ,,216kb,PromegaWizardDNAclean-upsystemkitTE(pH714)1215ng/Ll,PCRCre,DNADNAEcoRÕ018%Southern/0[9]2211Crepcol2A1-CreCre610kbÒ112kbCre211kbployA,1213kb(2)2pcol2A1-CreFig.2transgenicconstructpcol2A1-Cre212col2A1-Crepcol2A1-CreNotÑ,913kbÒCreployA3,TE2ng/Ll,344,32314,8,525711%,1611%(1)5210Cre,1912%3PCRCre1Table1ProductionoftransgenicmicebymicroinjectionNo.ofembryosNo.ofembryosNo.ofrecipientNo.ofpregnantNo.ofnewNo.oftransgenicmicroinjectedtransferredfemalemicemicebornmicefoundermice34432314852103PCRCreM:x174/HaeÓ,13531078872603310281/271234194bp;1~5&7~8:col2A1-Cre;6:;9:Cre;10:Fig.3IdentificationofCretransgenicfoundermicebyPCRM:x174/HaeÓ,maker,13531078872603310281/271234194bp;1~5&7~8:col2A1-Cretransgenicfoundermice;6:non-transgenicmouseasnegativecontrol;9:negativeCretransgenicmouse;10:transgenicconstructaspositivecontrol42629213Crecol2A1-CreCre,col2A1-Cre,LoxP(,Floxed/Floxed,,)PCRCreFloxed/Floxed,CreFloxed/+82LoxP(4)col2A1-CreCre,LoxP,LoxP,LoxP8Cre,P3P4,8,LoxP9234bpDNADNA,P3P4PCR,234bpDNA(4)8,CreCreDNA,PCR234bpDNAT-,LoxP9,PCRCreLoxPDNA4col2A1-CreCreM:100bpDNAladder;1:;2:;3:;4:;5:;6:;7:;8:;9:;10:Fig.4Crerecombinasewasexpressedspecificallyincartilagetissueofcol2A-lCretransgenicmouseM:100bpDNAladder;1:skeletalmuscle;2:cartilagetissue;3:intestine;4:skin;5:cardiacmuscle;6:thymusgland;7:lung;8:liver;9:kidney;10:non-transgenicmouseasnegativecontrolSouthernCre5,aEcoRÕ915kb,Floxed414kbCre,2LoxP,711kbPCRCreDNASouthern(5,b),711kb,Cre,col2A1-CreCre,LoxPSouthern-blot,Cre50%5col2A1-CreCreLoxP(a),LoxP,pSouthernP;(b)SouthernCoÓ-CreDNA,EcoRÕSouthern1:;2:;3:;4:;5:;6:;7:;8:Fig.5TheCrerecombinaseexpressedincartilagetissuecouldmediatedtherecombinationbetweenLoxPsites(a)Genomicstructureofthewildtypeandconditionalgenetargetesalleles.Rectangleindicatesexon,triangleindicatedLoxPsequence,pindicatesprobeforSouthern-blot.(b)theresultofSouthern-blot1:cardiacmuscle;2:liver;3:spleen;4:lung;5:kidney;6:cartilagetissue;7:intestine;8:brain5:Cre427Ò,3Cre,CreÒ,Ò[11,12]ÒATG,Ò[13,14],,,ÒCre,,Ovchinnikov[15]Sakai[16]Ò610kbCre,CreOvchinnikovCreSV40TpolyA,SakaiCreÒ52,Ò52polyACrepolyA,col2A-lCreCreCre70[17][18]Mx-cre,CreCre,CreLoxP,Smads[19],Smads,SmadsTGF-B:An-dersonBenoitdeCrombruggheÒA1;HuaGuCrepIC-Cre;Van-derbiltM.A.MagnusonployADNApBs-hGH[1]HoessRH,AbremskiK.Mechanismofstrandcleavageandex-changeintheCre-Loxsite-specificrecombinationsystem.J.Mil.B-iol.,1985,181:351~362.[2]Cohen-tannoudjiM,BabinetC.Beyond-knock-out.mice:newper-spectivesfortheprogrammedmodificationofthemammaliangenome.Mol.Hum.Reprod.,1998,4:929~938.[3]YangXJJ,LetterioRJetal.Targetedd