顺式调控元件的寻找20121116

分子生物学工作基础基因的表达调控刘喆[e-mail]3思考题：如何研究顺式调控元件的功能？4LuciferaseAssay基因敲除5LuciferaseAssay6LuciferaseAssay质粒构建细胞转染DualLuciferase分析24-48小时7细胞转染电转染磷酸钙转染脂质体转染（lipofectamine2000）8电转染细胞上短时间暂时性的穿孔让外源质粒进入9磷酸钙转染通过DNA，氯化钙与磷酸缓冲液混合，沉淀形成保护DNA且极小不溶的磷酸钙颗粒。这些磷酸钙-DNA复合物粘附到细胞膜并通过胞饮作用进入细胞中10脂质体转染膜融合的方法将外源DNA导入细胞11转染效率的矫正共转染：Firfly和Renilla萤火虫(Photinuspyralis)萤光素酶和海肾(Renillareniformis)萤光素酶Promoterandcisregulatoryelementsexon1Cisregulatoryelementenhancer,silencer,insulator,CGisland,MAR,DNAtetheringfragmentexon1CisregulatoryelementLuciferasePCisregulatoryelement14151617LuciferaseAssay的功能确定启动子位置判断顺式调控元件的功能检测转录因子是否能够直接调控基因的表达初步判断基因表达变化的原因18基因条件性敲除确定启动子位置判断顺式调控元件的功能检测转录因子是否能够直接调控基因的表达初步判断基因表达变化的原因19LoxP:ATAACTTCGTATAATGTATGCTATACGAAGTTAT顺式调控元件（Deletion）20ATAACTTCGTATAATGTATGCTATACGAAGTTATATAACTTCGTATAATGTATGCTATACGAAGTTAT思考题如何研究转录因子A是否调控基因B？如何调控基因B？DNA和蛋白质的相互作用EMSAChIPR.Voll09/01RegulatoryRegionCodingSequenceGeneRegulationbyTranscriptionFactorsApplication:R.Voll09/01DetectionofDNA-bindingfactors/proteins•AnalysisofDNAsequences(e.g.promoterorenhancerregions)fortheirpotentialtobindspecificallytoproteins/nuclearextracts•Analysisof(sub-)cellularextractsforthepresenceofcertainDNA-bindingproteins(e.g.atranscriptionfactorwithaknownrecognitionsequence)EMSA:PrincipleR.Voll09/01Adouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabeledwitharadioactiveisotopeandincubatedwithanuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.NF-BFreeProbeRadioaktivelylabeledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-BRadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe)Nuclearextractofnon-activatedcellsNuclearextractofactivatedcellsPreparationofNuclearandCytosolicExtractsR.Voll09/01Theprocedureiscarriedoutonicerspat4°Candinthepresenceofprotease(andphosphatase)inhibitors.1.Swellcellsinhypotoniclysisbuffer2.AddNP-40andvortextodisruptcytoplasmicmembrane3.Centrifugetopelletnuclei4.Carefullyremovesupernatant(containscytosolicandmembranefraction)4.Washnuclearpelletonceinlysisbuffer5.Addhypertonicextractionbuffertonuclearpellet6.Agitatevigoureslyfor30minutes7.Centrifugeathighspeed8.Removenuclearextract,determineproteinconcentrationandfreezeondryiceuntilEMSAisperformedTheProbeR.Voll09/01DoublestrandedradiolabeledoligonucleotidescontainingatranscriptionfactorbindingsiteAP-15’-GCTTGATGACTCAGCCGGAAC-3’3’-CGAACTACTGAGTCGGCCTTG-5’NF-B5’-AGTTGAGGGGACTTTCCCAGGC-3’3’-TCAACTCCCCTCAAAGGGTCCG-5’BindingmotifR.Voll09/01AnnealingtheOligosHeatupanequimolarmixtureofthe2oligosto95°Candletthemslowlycooldownbyturningofftheheatblock.LabelingtheProbeR.Voll09/01A.T4PolynucleotideKinase5’-AGTTGAGGGGACTTTCCCAGG-3’3’-CAACTCCCCTCAAAGGGTCCG-5’5’-P-AGTTGAGGGGACTTTCCCAGG-3’3’-CAACTCCCCTCAAAGGGTCCG-P-5’PNK+Adenosin-P-P-P(g-ATP)RemovaloffreeradioactivematerialR.Voll09/01RemovenotincorporatedisotopbySephadexG50columnReagentsR.Voll09/01CompetitorDNA:Competitionofunspecificpoly(dI-dC).poly(dI-dC)binding(e.g.histones)RadiolabeledProbe:DetectionofDNA-bindingproteinsReactionBufferBindingconditionsAnalysisbynon-DenaturingPolyacrylamideGelElectrophoresisR.Voll09/01准备非变性PAGE凝胶上样电泳干胶放射自显影ProofofSpecificityR.Voll09/01SupershiftusingantibodiesagainsttheDNA-bindingproteinCompetitionforbindingtotheradiolabeledprobeusingunlabeledwildtypeandmutatedoligosR.Voll09/01SupershiftAdouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabelledradioactiveandincubatedwithanuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.p50/p65FreeprobeRadioactivelabelledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-BRadioactivlabelledoligonucleotidewithNF-B-bindingsite(probe)NuclearextractofactivatedcellsNuclearextractofactivatedcellswithanti-p50antibodyp50/p65+anti-p50R.Voll09/01CompetitionwithUnlabeledOligosIncreasingamountsofunlabeledoligoscontainingtheNF-Bbindingsiteorunlabeledoligoswithamutatedbindingsitewereaddedtothereactionmixpriortogelelectrophoresis.Specificbindingisextinguishedonlybythenon-mutatedoligo.p50/p50Freeprobep50/p65UnspecificWildtypeoligoMutatedoligoGGGGACTTTCCCGGAGACTTTCCC