GENETIC ENGINEERING

41. 2. 333.3DNADNA4.

.3 1.60008000/1/10.8 2.195035 3.1973Cohen Groupterr+nersrterrner 3019972003

PscloltetrRb-3nersrtetrner tetr nerOtetrne8-1 Cohen Group

() 1.19 2. 3. 300/50/

() 1.TMDNMD() 2. 3.idea

gene DNARNAgenotypephenotypegenetic engineering DNADNA(DNA),

1. DNA 2. DNA 3. 4. IL2IL2/LAK

1.DNABerg 1972BergE.coRSV40DNAphageDNADNABergDNA 2.Cohen 1973Cohen GroupE.colitetrpsclolnersrR6-3E.coliterrNer,

1. 2. 3. 4. 5. 6.

() 1997223NaturePPL19972003/7/12 ?

1. 2. 3.

8-2

3331. 2040AveryDNAAvery102. 2050Watson-crickDNA3. 2060DNARNAPr,43

3 1 1204060dsDNA1042.2*1011 2 31970SmithWilcoxHaemophilus inffuenzaeHind

2() 1ligaseDNA DNA 21946LederbergF.50-60RCoE() 31973Cohen

31970BaltimoveTemin (1)HGP20052.813kb/1042.2*1011 2mRNAmRNA 3mRNAcDNA (complementory DNA) BergCohen

clone,cloning 1. 2.molecular cloning 3. 1 DNA1 genetype DNA 2DNADNA

DNA(DNA recombination technique) DNADNADNA Biologic engineering 1.genetic engineering 1DNADNA 2 3 4 5 23456

tool enzymes 1.resriction enzymes,restriction endonadease)2.DNADNA ligase,ligase)3.(reverse transcriptase)4.DNA(DNA polymerase,DNA pol)5.(nuclease)6.(terminal transferase)7.(BAPCIP)1. 2.

1. restriction endonuclease,restrction enzymesDNADNAdsDNArestriction=clearage=hydrolysis 2. 400E/350M3 1DNA 2 Mg2DNA

3. 1973Smith 3 2 EcoREscherichia coliR

() 1 4 1DNA 2dsDNA46bp46bp64 31800 GC CG GTNAC GAA TTC Hha CG GC Mae CANTG EcoR CTT AAG (4)DNA DNA 4bpHpa,MboDNA441/256 6bp461/4096

2.; GTYRAC Y=CT R=GA YR=CGCATGTA Hin 4 3.(),DNA Hga GACGC5/10()dsDNA 5 GACGC NNNNN NNNNN 3 3 CTGCG NNNNN NNNNN 5 Distant cleavage

() dsDNA 231 flushbluntend TCGA-smabsuR or TC3+5GA- AG CT-Alu -AG5+3CT-2.5 5cohesive end3 5-GAATTC3EcoR 5-G AATTC-3 + 3-CTTAAG5 3-CTTAA G-53.33cohesive end 5CTGCAG 3 pst G-3 5-CTGCA + 3GACGTC 5 ACGTC-5 3-G sticky end2

(isoschizomers) 1Isoschizomers: (1) Mbo 5SauS 3 NNGATCNN-3-NNCTAGNN-5 (2) SmaXma CCCGGG CCCGGG (3)* HpaCCGG MspCCG*G MboGATC Sau 3AGA*TC 2.subsets- Hpa CCGG Sma6bpHpa4bpHpa Sma CCCGGG Sma6bpHpaccgg6 Sma 3isoaudumers- BamH GGATCC DNASau 3A NGATCN DNA

()buffer18 1. 2. 3. 4.pH 5. 6. 7.2Mg2+Mn2+Co2+Zn2 8.DMSO PoliskyMgcl25mM2mMpH8.5EcoRI6bp4bpNAATN,AATTEcoRIEcoRI*DNA1212 1DNA 2.DNA 3.DNA 4.DNA 5.,DNA 7.DNA 8.

DNADNA

DNA DNAT4DNA ligase. dsDNA3OH5P T4EcoliMW.68KD 1ATPMg2+ 2NickDNA 31100

RNADNARNA dependent DNA polymerase,RDDPBaltimovemurine leukemia virus,MLVMizutan(Rous sarcoma virus,RSV)1970Natureretro virus mRNAcDNAcomplementory DNA AMVMc-MLV()

sscDNAEcoli DNApolkelnowcDNAcDNA 1. mRNAcDNA 2.DNADNApol ssDNA3OHDNA53DNA 3.RnaseH RNARNADNARNA 53RNA53RNA 35RNA35RNARnaseH 4.DNA Mn2cccDNA 5.DNA unwinding

DNA DNADNADNA DNAE.coli DNA pol 1956A.kornbergE.coli165MW109KD19451Zn2Zn23DNAdNTP 1E.coli DNA pol353DNA3553 153DNA ssDNA3OH53 5CCGATAOH E.coli DNA pol 5CCGATAGCCT 3 3GGCTATCGGA5 Mg2dDNP 3GGCTCGGA 5 2 35 3OHssDNAdsDNADNAdsDNA53 5CGCATCG-OH 3 E.coli DNA pol 5CGC 3 GCG Mg2 3 GCG dAdTdCdG dsDNA53DNA 353 5dsDNADNA:RNARNARnaseH5CTCATTAG3 E.coli DNA pol 5CATTAG3GCGTAATC5 Mg2 3GCGTAATC +pC,pG

2. E.coli DNA pol(DNA pol) 1 DNA polDNANick translation32P6400.00/ 53 35 dsDNA Mg2 DNA 5 3 3532P dNTP,DNA pol 53 35 2 DNA polDNAGapped regionDNA cccdsDNA DNA1ssDNA DNA pol ligase(3-OH,P-5)DNA

3DNA DNA pol3SangerSaugerDNA polssDNADNA Sauger DNA pol23ddNTPPAGEDNA Mg2Mn2DNA polNTPdNTPNTP1NTPDNA dNTPDNA polT4DNA pol44dNTP1dNTPdNTP1dATPDNA pol 35A33dNTP8DNA

DNAKlenow E.coli DNA pol1KlenowE.coli DNA pol 1.KlenowMW76KDE.coli pol 53DNA35 2. Klenow (1)dsDNA53 5G-OH Klenow, Mg2 5GAATT 3CTTAA Dntp 3CTTAA (2)132p dNTPDNA3 (3)cDNAcDNA (4)Sanger ddNTP

T7DNA: 1.T7DNA 1 T7E.coliDNApolT7 2 Klenow533553 53 53DNADNA pol 35 Klenow1000Klenow3DNA 2 T7DNA polSangerDNA

TaqDNA(Taq DNA polTaq pol DNADNA53537580,37 10PCRpolymerase chain reaction 1. 1969thermus aquaticus strain,TYC7075cetusTaq DNA polymerase94KD206Taq DNA polymerase94953040PCRVENTsacTaq

2.Taq pol5PCR 1 PCR cycleDNA3060301530Taq pol30 2 Taq pol755570Taq pol 3 PCR>1kbDNApol>250bpKlenowTaq pol2kb

4 DNA polTaq pol251107Klenow5 DNApolPCRDNA Klenow1/10000,Taq pol1/5000Taq pol25400 T4DNA pol

T4DNAT4 DNA pol 1. T4E.coli 2. KlenowKlenow 153 235 T4 pol/Klenow 3.Klenow53DNA3533 5CTGCCTGCA3 T4DNA pol 5CTG 5-CTACC 3-GACGG 32P-dCtp,dNtp 3GACGG 3-GACGG

SISI,nuclease SISIBAL31SI(SInuclease SISI) 1. aspergillusoryzas 2. ssDNAssRNAdsDNADNA-RNA 3.3 (1)ssDNAssRNA5pDNA>RNA,ssDNAssRNA SIPH4,5 Zn2 5pdN 5prN (2)SINicksmall gapsdsDNA (3)SIdsDNADNA-RNASISI DNA cDNA DNARNADNAmRNARNAloopSI

()BAL31 1. 2. 3 3. 13dsDNAdsDNA3OHssDNAdsRNA 2 DNADNA 4 1dsDNA 2 3DNAzDNAb-DNA

TTE DNApoldNTPDNA3OHterminal transferaseTTE 3OHssDNA3-OHdsDNAdsDNA 1. dNTP>DNA3OH ssDNAOH TTEMg2Mn2 DNApdNnppi ndNTP 2. 3OHdsDNACo2 dsDNA TTE Co2 DNApdNnnppi ndNTP 1>cDNA> 2.32pDNA3

alkaline phoptase, APE APEBAPCIPDNARNAdNTP55-PDNA32p5DNARNA5 -P

1.(restriction endonuclease,restrction enzymes400RE/350M

dsDNAMg2dsDNA,5-3-1DNA2.3.4.5.DNA6.2. ligaseT4phagedsDNAMg2+ATP>1DNA2.DNA3. DNADNA polE.coli533-55-3ssDNAdsDNAdsDNAssDNAMg2dsDNA53pol1.2.DNA

4. ReverseTranscriptionRNARNAcDNADNADNARNAseHssRNAssDNARNA:DNAcccDNAMg2RNADNAcDNA5. SINase SIssDNAssRNAZn2DNA6. Bal31dsDNACa2537. TTEdNTPDNA3OHdsDNA or ssDNA3-OHMg2Co23OHDNA32pDNA38. BIP/CIP/ds,ssCa2Mg2

1.vector 2.DNADNAvector 3.vectorDNADNA

1.12 PBR322PCDN3

2.123PUC8PBUDCE41YE3.4.(1)

1.sbuttle vector YEP,DIDB2192.YAC Yeast Artificial Chromsome PBR322HGP3.BAC

3 DNAMCSDNA/

1.cloning vector DNA 2.Expression vector ori,Ampr,Mcs/DNASDsRbS

ori Amp Mcs E.coli P Rbs/SDs Mcs terms P/E Mcs terms polyA

Ecoli1. Ori 2. Ampr 3. MCS 4. /1. Ori2.Ampr3.Mcs4. Promoter5. SDs Rbs 6.Terms7.E.coli 1.orip E.coli2.Ampr 3.Kanr 4.Orie 5.Mcs 6. P/E /7. Terms 8. polyA9. 78

4 (1) /: (2) (3) // (4) P

1.Orioriginori123ori425 DNAE.coli 10200 DDDPI 10003000 PBMI

2. genotype phenotype1Ampr 2tetr 1 3camr 4neorkanr G418 5hygr

3. MCSmultiple cloining siteMCS 1MCS DNA//DNA 2polyliker 3linker bpdsDNA (2)3likeradaptor

4pormoter 1 DNADNA DNAupstreamRNApol DNARNApol DNARNApol3

2downstreammRNAdsDNARNA

E.coli4060bp35bp2TATATFDTATAboxRNApol10bpTATA25bpATTATAHognessRNApol120bp1bpRNAbp

3

1. Ecoli1Lacuv5uv52trp3Tac4PLphageCI857ts2. 123. 1SV40 (2)3Melallothionen4. 1210K5. 12Napalin3Tubulin

LacLacuv5TrpTacPLT7

1LacE.coli1Pg2CAP3O4SCAPcAMP>P>RR>O>>>>>2Lacuv5IPIG>>O>CAPcAMP>LacIPTG50

3TrpE.coli1Pg2R3O4SETrpTrp>>Trp>Trp>O>Trp>>508104TacTrpLacTrpLac1tac=Trp-35+ Lacuv5-202tacTrp-3546bppribrowLacO3tac12Trp35Lac10OSDIPIGLac1Lacuv572RB791XLblueSB221JM109

5T7Lacuv5T7RNA polIPIG1Lacuv572RB791XLblueSB221JM109

5./enhancerE/silencernegatire enhancer 1 / 2 535100300bp 3 DNA>E/P DNA>topDNA RNA pol

6. //SDRbs/AGU/SDs mRNAAUGSDAUG311bp39bp16sRNARNAJ.shineL Dagarne1974 mRNA 5-----AGGAGGUUGACCUAUG---------- 3----AUCCUCCUAG,----------- rRNA31mRNA/RbsRbsE.coli2pr.SDAUGSDAUG

7/terminator o transcritionterm terminator of translation, terminator codon,stop codon 1/term down streamDNARNA polmRNAtermIR 700800bp 2/terminator codon mRNA3UAAUAGUAGnonsense mutation supperessor

8polyA AATAAAdownstreamGTT2polyA mRNAAAUAAAGUURNApolAAUAAA1030bpRNApolyA 78/,3 33poly(A)

DNA 31 DNA2 DNA3 downstream techniques of GEDNA1 2 3 DNA4 DNA5 6 7 8 /9

(xbal I) EcoRIEcoRI (Hind III) PUC(xbalI)EcoRI(Hind III)EcoRIEcoRI+EcoRIEcoRIEcoRIEcoRIDigestion with EcoRIRecovery of APPAI DNA fram agaroseAPOAIPUCQ-APOAIPBR325-APOAIPUC9 PUC9APOAIEcoRIEcoRIEcoRIEcoRIFigure8-3 strategy of construction of recombinant pucq-APOAI plasmiaE.coli

Preparation of PBR325APOAI and PHCo plasmidDigestion of PBR325-APOAI with EcoRIDiestion of Puco with EcoRIIsolation and Recovery of APOAI gene fragmentLabeling of DNA probe with isotope or biotinLigationTransformation of EcoliSelection of TransformantsIdentification of plasmid plasmid by enzyme digestionIdentification of recombinatn DNA by southern hybridizationFigure8-4 The experimental process of the construction of recombinant DNA

/// 3 1.DNA// 2. 1

33 4 3.MCS 4 11.5kb 10

cccDNA 1.> 2.>DNA,RNA, 3.>DNAMCSDNA DNA //2000.0010000.00////> //

1. cDNA 2. 3. DNAcDNA

1. 1DNA DNA DNADNA DNADNA

2 DNAchromosome walking DNA DNA southern blot , DNA3DNA

2. PCRRT/PCR1 / PCRDNA/RT-PCR(mRNA) 2 RT/PCR mRNApolyA mRNAcDNAcDNA cDNA PCRcDNA3.GenBank

cDNA 1 DNA cDNA mRNAcDNAcDNAcDNA 2cDNA4.DNA

DNA DNA DNA bus DNAgenotypephenotype DNAT4DNA ligase

1. / ligaseDNA gene EcoR vector EcoR 1 > DNA31APBIP/CIP5P 2/ 53

2. 1/2 EcoRPstE.coR gene Pst EcoR vector Pst3./ 5 G GATCC3 Saw3AI gene BamHI vector 3 CCTAG G 5 ligase

(xbal I) EcoRIEcoRI (Hind III) PUC(xbalI)EcoRI(Hind III)EcoRIEcoRI+EcoRIEcoRIEcoRIEcoRIDigestion with EcoRIRecovery of APPAI DNA fram agaroseAPOAIPUCQ-APOAIPBR325-APOAIPUC9 PUC9APOAIEcoRIEcoRIEcoRIEcoRIFigure8-3 strategy of construction of recombinant pucq-APOAI plasmiaE.coli

4HaecDNADNAcDNAcDNA/vector

cDNAcDNAcDNA/vector533cDNAcDNA(cDNA/vector)TPCRT/A

PCR

*4.13DNApol3OH53 25 5 3 3 5 3 5 5 3 3 EcoR 5GATCGAATTC3 5GATCG-----

1.

DNA EcoR linker5P PGGAATTCC GGAATTCCCCTTAAGG CCTTAA GGPT4ligase GGAATTCCGGAATTCC CCTTAAGGCCTTAAGGGGAATTCCGGAATTCC--- CCTTAAGGCCTTAAGG---

EcoRI AATTCC GG GG CCTTAAEcoR 2. linkerEcoRlinker

* 1linkerEcoR 1linkerEcoR (2)DNAlinkerSal/Not100/1000bp1 3adaptorlinker

3.adaptorlinker adaptorcDNAlinkercDNA Linkeradaptor adaptor gene adaptor

vector

4.dCdGDNAcDNA 15CTGCAG pstI Pst3OHTdTdC(3GACGTC 2 Pst3TdTdG 3/cDNA 4rDNAcDNAPstcDNA

5.TA /T PCRcDNA1cDNAAA Taq polPCR3AADNA pol2TTT4 MbcXcmHphT TdTT TaqpolTTAA T

DNArDNA1. 1 rDNADNA 2 rDNA2 1 2 2. (1)rDNA(2)(3)DNA3. transformation/transfection rDNAtransformation transfection transduction DNA

rDNA

1. DNAE.coli(1) CaCl2Ecoli (2)rDNA>>42>rDNAEcoli (3) >1compent cellsDNA2elecroporationDNAEcoli2. phagerDNAEcoli(1) phageDNADNAEcoli(2)(1)Ecoli(2)rDNAEcoli(3)3. cosmidEcoli(1) cosmidDNArDNA(2) Ecoli(3)rDNA2cosmid

E.coli E.coli 8-3DNA hostrDNADNA

xgalPucPUC18/19E.coli DNA (1) Lacz(N)N 146AA (2)MCSAA N (3)PlacLacz pr.IPTGJM103lacz(c) C JM103 lacz(n)lacz(c) prX-gal543MCSgene

PUC19PlacLacZ(N)MCSJM 103 IPTGlacz(c) X-gal-8-5 -

DNA 1.PBR322 Amprtetr,BamHISalI 2Amprtetr 2.tetrtetr 3. PBR322AmptetAmpr 4.tet2Amptet

PBR322AmprterrBamHIsal I8-6

1. DNARNADNA/DNADNA/RNADNA 2. 3

DNASouthern blotDNADNARNA1. 2.RNANorthern blotRNARNADNA3. 4. Western blot 5..

3. (Hybridization in site) 1975Granstein Hogness ,southern blot .() DNA.DNA,..DNAcDNA,.: NC LB, DNADNA,,,X

: (5-) DNA().: . rDNADNA. EcoRIPBR325-APOAIPUC19-APOAI2.7kb5.4kb0.89kb8-7 PUC19-APCAIPBR325-APOAIPNAECORIPUC19

4:

.DNA DNADNA.,,,,.DNA,,. ,: 1., DNA,, 2.DNA,( 3.()()3 ,,,rDNADNA,,

: 1. 2., ,: 1., 2., 3. 4. ,,,

4: ,4,

1.RNApolpromotor,.2.mRNASD,rRNA,.3.,,mRNA, ;4.,.1promotorSDs,,SDsrRNA,.1,2.3,4 mRNAcDNA,cDNA,,,,N,,.

1.()2.mRNAmRNA3.Pol rRNA,mRNA, t RNA, 1pol()4.TATA(Hogens)box (-12~~-32) CAAT box(-70~~-80)TATA(pribnow) box (-10~~-35)5.3AAUAAA6.5m7GAUG3-12bpSD

\ 4 E.coli ,: 1.; 2. 3.

(E.coli)

E.coli5E.coli33

E.coli5E.coli3E.coli31.,E.coli;2.;3.promotor;4.mRNA,;5.MCS.1.,;2.,,, E.coli;3., E.coli,.1. ,,,;2. , .3. ,,,,

E.coli

1cDNA1cDNA5cDNA2cDNAmRNA2P/SDSE.coliE.coliRNApolP/SQSPLTacT73/4PLCIts85T7T7RNApolPL300C400C420CT7IPTG

33 1. /1. DNAmRNAcDNA2. PCR/RT/PCR/2. DNA3. 3.

()

41///1TPEG()Lacz()2GST/ompApelBphoAtagHSP,E.coli GroEL,GroES,Dank,HTPG2IP/SD5;343()54///6

()/

LinkerAdaptorcDNATPCRT/A

() 1 PL(Its85) T7T7RNApol 2 PL300C400C420C T7IPTG PL ,T710702

3.1PLpPLM52/2T7PET11JM103(DE(1)CIts857280C300CIts857PL400C420CIts857T7IPTGT7RNApolIPTGT7RNApolT7IPTGT7RNApolT7(2)OriAmpPLNHpaItermNHpaOriAmpT7SDSPMCStermSP(3)DNACIts857DNADNALacllv5T7RNApolDNA(4) E.coliCIts857 PLHSP

()(fusion protein) DNAcDNANDNACcDNA ()5 / E.coli

() 1reading frame mRNA3AA31AUGAUG GCA AAA UUU CCCAUG/GCA/AAA/UUU/CCC/, A/UGG/CAA 23 1 53NC

3AUG 5PSDAUGPeptidegeneMCS

4/cDNA 1.2.3Charnay1978TaconTalmage19803/1pPC1pPC2pPC3rDNA3 3pPC1GAATTCG NN/NNN/NNN/CTTAAG (NNN)n1(NNN)n+N1(NNN)n+2N pPC2GGGGAATTCGGG NNN/NNN/NNN/ CTTAAG(NNN)n pPC3GGGGGAATTCGG N/NNN/NNN/ CCCCCTTAAGNNNn+2N(1)(2)2

LaCzPUR/PEX123/ORF (1) PUR823(1)PUR278LACI/TGT/CAA/GGGG(2)PUR288 (BamHI)LACI/TGT/CGG/GGAGGA(3)PUR289LACI/TGT/CAG/GGGGG

(2) IL2 1. IL2IL2 2.PCRIL2 3.PUC18IL2 4.PUR/IL 5.DH5 6. 7. 8.(NH4)2SO4 9. 10.

5>SDs>ATG>>>3 NdeINcoIATG SDs1NdeINcoI>>NdeINcoIATG1.2.Inclusion body(1)>50%

25 5P SD AUG Signal peptide MCS cDNA 3Ecoli2 1. 2.

1.SDS-PAGE PI2.Western blot

Ecoli 54

41.1.2.2.mRNA3.P/SDs3.4.4.5. 4

12SDAUG3MCS()45 6 7

mRNATNF-aIFN AUGSDs 1SSSuAAGAGG >AAGGA 36 2AUGuAAAGGAGG5bp AUGSDATmRNA

1(2)IPTGPL

T7

Tac30Cits85742IPTGT7RNA polLac Trpp

1. 11AUG6 tRNA tRNA2. Ecoli8

ArgAGA/AGG/CGA/CGGGlyGGA/GGGSerUCA/ACG/UCG/UCCLeuCUA/CUCPrcCCC/CCU/CCAILeAuGCYSGGA/GGGThrACG

3. 2EcoliE.coliCmRNAAUA/AGA/AGG3%-4%11%-14%tRNAE.colitRNAtRNAArg(AGG/AGA)AGG/AGAProuktPAE.coliE.coli tRNAArg(AGG/AGA)arg(dnay)

mRNA E.colimRNAmRNA/mRNA2: 1.mRNA5 E.coliomPAmRNA5mRNA5mRNA 2.mRNA E.colirepetitive extragenic palindromic, REPCat3

NPhe,Leu,Asp,Lys,Ang

1.PL CIts857 32CIPL 42CIPL 2. Tac LacI LacILac> IPTGLacO

() HSP()31. 2. O213. OD600=0.6,

1.lon-E.colilon,lon->>2.rPOH-RopH3. PinPinpinE.coli

(E.coli)

E.coli0-60%0-10%0-300-1%+/-+/-++-++++/-++++/-+/-++-+++-+++--++

E.coli E.coli1.2.3.

()() 1. 2. > 3.

100 2Virus infection Transfection

1

2 DEAE

1. 2. 3. 4. (Pnasterone)

DNA DNA4872

1mRNASouthern blot

2PAGEWestern blot3>>

2 1. : (1)(2)(3)(4)(Ampr)

1CMV1EFlSV40early1

EF-1140160CMV4RSVLTR2SV40 later401.1SV40 early401Adeno major late0.4-globin-0.2

2 P0ly A11 30 AAUAAA GU- U- Poly A mRNA Poly A UPoly APoly A103

Poly A BGH3SV40 late402TK15SV40 eary401Hep B1

3104 APHneorgellticin418)APHneorG418neoG418G418G4 18100mmolLHEPESpH4100 G418

ADA ADAadenosine dearminase ADAXylAxlofiuanosyl adenine Xyl ATPADA 10g/ml15g/ml,4mol/Lyl-A0.010.3mol/L 2 deoxyconformycindCF dCF ADAANAdCF 13 kDa bleomyclnphleomycinzeocinbleomyclnphleomycinzeocin0.1gml HPH HPH Bhygromycin B phosphoptransferaseHPH hygromycin B 10400 gml Hygromycin B HPH Hygromycin BHygromycin B

4 1982,Gormanchiomphenicol acetyltlansferaseCATCATmRNACAT 5epitope tagsGSTIgGlFcFLAGDykDDDDKHAYPYDVPDYAHis6HHHHHH C mycEQKLISEEDL

2 1pcDNA3.l Invitrogen8-7CMV(BGH)GFPHismycHisGXpressV5Hyromycinzeocin8-7 pcDNA3.1

2pSIPromegaSV40SV408-88-8 pSI

3pCMV-HApCMV- HACLontechCMV CMVSV40 19 S mRNA SV40 16 S mRNANHA8-9

8-9 pCMV-HA

4pBudCE4.1 Invitrogen CMV EFl8-10

8-10 pBudCE4.1

5pTREpTREpTetOffpTetOnTetDoxClontechpTetoffpTetOnetRHSVVP 16tTArtTApTRETRETetOffTetDOXpTettTApTRETREtDoXtTATRETetOn,PTetOn rtTA tTA TetR Tet Dox TRETet Dox TetDoxrtTATRE8-11

retrovirusadevirusadenoassociated vimsVaccinia vims baculovirus 1 RNADNA

ClontechpLNCX 8-12pLNCXpLNCX M01oneymurine leukelnia virus Moloneymurine sarvoma viruspBR322 pBR322ColE1 ampr53 1ong terminal repeats, R;5LTR+ Hygromycin BPCMVIEackaging cell line Hygnmycin B

2 Clontech pAdenoX Vial DNAPtre-ShuttleQiagenpCR259pCR276adenoviruspAdenoXDNA8-13PAdenoXDNA

8-13 pAdeno-X

1 1 2 3N 4 5CATluciferase

2 NC 6HIS GSTNCNCgreen fluorescent protein GFP 3 DNA

4 5 DNA

ATCCAmerican Tissue and Cell Collection 1.L-Ltk-NIH 3T3 Sp20 NSO 2.HEK293HeLa HL60HT1080CHOCHOK1BHK

1. 2.COS HEK 293 COS CV l 3. SV40 SV40 SV40 THE K293human embryonic kidney2935DNAE1A CMV

1. 2.globinHelaMELMEL 3.CHOCOSHEK 293

DNADNARNA DEAE 1.DEAE- DEAE-,DEAE

2. DNAPBSDNADNA 3. DNARNADNARNADNADNA InvitrogenPromegaClontechQisgen

Ca2+Mg2+PBS HeBS137 mmolL NaCI5 mmolL KCI0.7mmolL Na2HPO4 6mmolL21 mmolL HEPES pH7.05 272 mmol/L7 mmol/L K2PO4(pH7.4)l mmol/MgCl2 1.eleetroPoration 1982Whng

DNA(1 20g10-40g) DNA10l TE 5075 4 640g5 min 124 640g 5min,1107ml 8107ml

Cuvette 0.5ml DNA 5 min250-750Vcm25F20-100 ms10min2048h 48 72 h

2. 3. DNADNARNACSCIDNADNAA260nmA280nm1.81.9DNATEDNA1mgmlDNADNADNA

DNA PCR PCR PCRPCRTal DNA polPCRabDNAabDNAabcdbcPCR5533DNA poldNTP3OHadPCR5PCR

1 bcad2PCRPCR DNTP50200mol/L TaqTaq3 centricon100 /4adPCR

abPCRcdPCR dsDNASSDNAssDNA dsDNA PCR-PCR33Tag pol

abcdDNADNAbcPCRDNA8-15

1. cDNA 2. () vit() 3. : . ,