基因表達與調控基因表達與調控1

研究基因轉錄調控的方法ReportergeneassayEMSA(Electrophoreticmobilityshiftassay)Foot-pringtingMethylationassayTet/offandTet/onassayNorthernBlottingChIPSAGEWesternblottingassay研究基因轉錄調控的方法2ChromatinImmunoprecipitation(CHIP)----real-timeanalysisforprotein/DNAinteractionYoumustknoworhave;1.DNAsequencearoundinterestingbindsites2.Proteinsthatcouldbindtothesites3.AntibodyfortheproteinChromatinImmunoprecip3Example:RNAPolIIattranscriptionstartpoint---NucleicAcidsResearch,2004,32(11):1-8ObtaintissuesfromanimalorhumanCrosslinkchromatinandbindingproteinsTreattissuesby1%formaldehydefor12minandthenstopreactionby0.125MGlycineDisaggregatetissuebyhomogenizer,collectcellsBreakcellsbybuffercontaining0.5%NP-40,spintocollectnucleiBreaknucleibybuffercontaining1%SDSSonicatechromatininto~500bpfragmentsBlockproteinA/GagarosebylDNA,tRNAandBSAExample:RNAPolIIattr4IncubatesonicatedDNAwithblockedproteinA/Gagarosetoremovenon-specificbind,spintogetsupernatant,savesampleas“Input”Incubatepre-cleanedsupernatantwithanti-RNAPolIIantibodyAddblockedproteinA/GagaroseintoreactionSpintocollectproteinA/GagaroseandwashIncubatesonicatedDNAwithbl5ElutechromatinDNAby1%SDS,100mMNaHSO3IncubatetoreversecrosslinkandusethesampleasPCRtemperateDesignPCRprimersthatflackthebindingsite(200~500bp)15.Rea-timePCRElutechromatinDNAby1%SDS,6基因表達調控課件7

SerialAnalysisofGeneExpression(SAGE)

----分析特定細胞或組織中的基因表達譜SerialAnalysisofGeneE8SAGEisbasedonthefollowingprinciple:9bpDNAtagcontainssufficientinformationtouniquelyidentifyamRNANNNN44=256NNNNN45=1024NNNNNN46=4096NNNNNNN47=16376NNNNNNNN48=65504NNNNNNNNN49=262016基因表達調控課件9BsmF1:GGGACNNNNNNNNNNNNNNNNNNNNCCCTGNNNNNNNNNNNNNNNNNNNNNlaIII:NNNNCATGNNNNNNNNNGTACNNNNNStreptavidin:AproteinproducedbythebacteriumStreptomycesavidinii.Ithasfourbindingsitesforbiotin.Ithasbeenusedextensivelyasprobesinimmunochemicalsystems,conjugatedtoantibodies,enzymesorfluorochromes.BsmF1:10SynthesizedoublestrandcDNAfrommRNAusedbiotinylatedOligodTprimer2.CutcDNAbyNlaIIIandmost3-endportionwasisolatedbybindingtostreptavidinbeads

3.dividecDNAinhalf(AandB)SynthesizedoublestrandcDNA114.LigateadaptorAorBtoeachhalfofcDNA4.LigateadaptorAorBtoea125.CutDNAbyBsmF1andbluntendsT4DNApolymerase

6.LigateAandBandamplifyditagbyPCRwithadaptorspercificprimers5.CutDNAbyBsmF1andblunt137.CutPCRproductbyNlaIIIandpurifydi-tag

8.Ligatetoconcatenation

9.CloneintoSph1siteinpSL301NNNNNNGCATGCNNNNNNNNNNNNNNCGTACGNNNNNNNN10.DNAsequencing7.CutPCRproductbyNlaIII14基因表達調控課件15

BertVogelsteinBertVo16

Tet-Off/Tet-On

Tetracycline(Doxycycine)controlledgeneexpressionsystemTe17

Background:GeneStructureofTn10OperonTetR:repressorfortetA,canbindwithTetracyclineT:Transposase–criticalforoperonjumpingRe:Resolvase–torelaxationoftwistsinDNAhelixTetA:Activelyexpelintracellulartetracyclineoutofthecell

18TRE:Tetracycline-responseelement

inthepromoteroftetA5`-TCCCTATCAGTGATAGAGA-3`,bindwithTetRtTA:Tetracycline-controlledTrans-ActivatorarecombinantproteinbyTetR(1-207Aa)—VP16C-terminalactivatingdomain(127Aa)rtTA:

BaseonthereverseTetR(rTetR)whichhas4aminoacidmutatescompareswithwildtypeTetRrTetR(1-207Aa)—VP16C-terminalactivatingdomain(127Aa)TRE:19TREinretrovirusplasmidpRev/TRE7XTREResponse

virusTREinretrovirusplasmidpRev20tTAinpRevTet-offrtTAinpRevTet-onRegulationvirustTAinpRevTet-off21

Tetracycline

DoxycyclineTetracyclineDoxycycline22

Tetracycline

DoxycyclineTetracyclineDoxycycline23基因表達調控課件24

Tet-off/Tet-onprotocolsMakeresponseretroviruspRevTRE-XCloneyourgeneofinterestintopRevTRE

Targetgene

25

Tet-off/Tet-onprotocolsTransfectpRevTRE-X,pRevTet-off/pRevTet-onintopackagingcells(PT67)separatelyResponsevirusandregulateviruslackviralgenegag,polandenvthatcanbesuppliedbypackagingcells

PT67PackageCells

3differentviralparticles

26

Tet-off/Tet-onprotocolsCollectviralparticlesfromculturemediumIfTet-off:Infecttargetcellsby:pRevTet-offviral+pRevTRE-Xviral;SelectcellsbyG418/Hyg;Culturecellsinthemediumwithdoxycycline;Beforeexperiment,removedoxycycline.

27

Tet-off/Tet-onprotocols5.IfTet-on:Infecttargetcellsby:pRevTet-onviral+pRevTRE-Xviral;SelectcellsbyG418/Hyg;Culturecellsinthemediumwithoutdoxycycline;Beforeexperiment,adddoxycycline.

28Determiningexpressionoftargetgene:AssayformRNA---PCR,NorthernblottingforProtein--enzymeactivity,Westernblotting7.Determiningeffectsofexpressionofinterestgeneonwhateveryouwanttostudy.Determiningexpressionoftarg29

基因表達與調控基因表達與調控30

研究基因轉錄調控的方法ReportergeneassayEMSA(Electrophoreticmobilityshiftassay)Foot-pringtingMethylationassayTet/offandTet/onassayNorthernBlottingChIPSAGEWesternblottingassay研究基因轉錄調控的方法31ChromatinImmunoprecipitation(CHIP)----real-timeanalysisforprotein/DNAinteractionYoumustknoworhave;1.DNAsequencearoundinterestingbindsites2.Proteinsthatcouldbindtothesites3.AntibodyfortheproteinChromatinImmunoprecip32Example:RNAPolIIattranscriptionstartpoint---NucleicAcidsResearch,2004,32(11):1-8ObtaintissuesfromanimalorhumanCrosslinkchromatinandbindingproteinsTreattissuesby1%formaldehydefor12minandthenstopreactionby0.125MGlycineDisaggregatetissuebyhomogenizer,collectcellsBreakcellsbybuffercontaining0.5%NP-40,spintocollectnucleiBreaknucleibybuffercontaining1%SDSSonicatechromatininto~500bpfragmentsBlockproteinA/GagarosebylDNA,tRNAandBSAExample:RNAPolIIattr33IncubatesonicatedDNAwithblockedproteinA/Gagarosetoremovenon-specificbind,spintogetsupernatant,savesampleas“Input”Incubatepre-cleanedsupernatantwithanti-RNAPolIIantibodyAddblockedproteinA/GagaroseintoreactionSpintocollectproteinA/GagaroseandwashIncubatesonicatedDNAwithbl34ElutechromatinDNAby1%SDS,100mMNaHSO3IncubatetoreversecrosslinkandusethesampleasPCRtemperateDesignPCRprimersthatflackthebindingsite(200~500bp)15.Rea-timePCRElutechromatinDNAby1%SDS,35基因表達調控課件36

SerialAnalysisofGeneExpression(SAGE)

----分析特定細胞或組織中的基因表達譜SerialAnalysisofGeneE37SAGEisbasedonthefollowingprinciple:9bpDNAtagcontainssufficientinformationtouniquelyidentifyamRNANNNN44=256NNNNN45=1024NNNNNN46=4096NNNNNNN47=16376NNNNNNNN48=65504NNNNNNNNN49=262016基因表達調控課件38BsmF1:GGGACNNNNNNNNNNNNNNNNNNNNCCCTGNNNNNNNNNNNNNNNNNNNNNlaIII:NNNNCATGNNNNNNNNNGTACNNNNNStreptavidin:AproteinproducedbythebacteriumStreptomycesavidinii.Ithasfourbindingsitesforbiotin.Ithasbeenusedextensivelyasprobesinimmunochemicalsystems,conjugatedtoantibodies,enzymesorfluorochromes.BsmF1:39SynthesizedoublestrandcDNAfrommRNAusedbiotinylatedOligodTprimer2.CutcDNAbyNlaIIIandmost3-endportionwasisolatedbybindingtostreptavidinbeads

3.dividecDNAinhalf(AandB)SynthesizedoublestrandcDNA404.LigateadaptorAorBtoeachhalfofcDNA4.LigateadaptorAorBtoea415.CutDNAbyBsmF1andbluntendsT4DNApolymerase

6.LigateAandBandamplifyditagbyPCRwithadaptorspercificprimers5.CutDNAbyBsmF1andblunt427.CutPCRproductbyNlaIIIandpurifydi-tag

8.Ligatetoconcatenation

9.CloneintoSph1siteinpSL301NNNNNNGCATGCNNNNNNNNNNNNNNCGTACGNNNNNNNN10.DNAsequencing7.CutPCRproductbyNlaIII43基因表達調控課件44

BertVogelsteinBertVo45

Tet-Off/Tet-On

Tetracycline(Doxycycine)controlledgeneexpressionsystemTe46

Background:GeneStructureofTn10OperonTetR:repressorfortetA,canbindwithTetracyclineT:Transposase–criticalforoperonjumpingRe:Resolvase–torelaxationoftwistsinDNAhelixTetA:Activelyexpelintracellulartetracyclineoutofthecell

47TRE:Tetracycline-responseelement

inthepromoteroftetA5`-TCCCTATCAGTGATAGAGA-3`,bindwithTetRtTA:Tetracycline-controlledTrans-ActivatorarecombinantproteinbyTetR(1-207Aa)—VP16C-terminalactivatingdomain(127Aa)rtTA:

BaseonthereverseTetR(rTetR)whichhas4aminoacidmutatescompareswithwildtypeTetRrTetR(1-207Aa)—VP16C-terminalactivatingdomain(127Aa)TRE:48TREinretrovirusplasmidpRev/TRE7XTREResponse

virusTREinretrovirusplasmidpRev49tTAinpRevTet-offrtTAinpRevTet-onRegulationvirustTAinpRevTet-off50

Tetracycline

DoxycyclineTetracyclineDoxycycline51

Tetracycline

DoxycyclineTetracyclineDoxycycline52基因表達調控課件53

Tet-off/Tet-onprotocolsMakeresponseretroviruspRevTRE-XCloneyourgeneofinterestintopRevTRE

Targetgene

54

Tet-off/Tet-o