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基因表达与调控基因表达与调控1
研究基因转录调控的方法ReportergeneassayEMSA(Electrophoreticmobilityshiftassay)Foot-pringtingMethylationassayTet/offandTet/onassayNorthernBlottingChIPSAGEWesternblottingassay研究基因转录调控的方法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基因表达调控课件7
SerialAnalysisofGeneExpression(SAGE)
----分析特定细胞或组织中的基因表达谱SerialAnalysisofGeneE8SAGEisbasedonthefollowingprinciple:9bpDNAtagcontainssufficientinformationtouniquelyidentifyamRNANNNN44=256NNNNN45=1024NNNNNN46=4096NNNNNNN47=16376NNNNNNNN48=65504NNNNNNNNN49=262016基因表达调控课件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基因表达调控课件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基因表达调控课件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
基因表达与调控基因表达与调控30
研究基因转录调控的方法ReportergeneassayEMSA(Electrophoreticmobilityshiftassay)Foot-pringtingMethylationassayTet/offandTet/onassayNorthernBlottingChIPSAGEWesternblottingassay研究基因转录调控的方法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基因表达调控课件36
SerialAnalysisofGeneExpression(SAGE)
----分析特定细胞或组织中的基因表达谱SerialAnalysisofGeneE37SAGEisbasedonthefollowingprinciple:9bpDNAtagcontainssufficientinformationtouniquelyidentifyamRNANNNN44=256NNNNN45=1024NNNNNN46=4096NNNNNNN47=16376NNNNNNNN48=65504NNNNNNNNN49=262016基因表达调控课件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基因表达调控课件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基因表达调控课件53
Tet-off/Tet-onprotocolsMakeresponseretroviruspRevTRE-XCloneyourgeneofinterestintopRevTRE
Targetgene
54
Tet-off/Tet-o
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