Description:
NFkBResponsiveLuciferaseReporterMCF-7StableCellLineisderivedfromhumanbreastcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNFkB responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation ofNFkBReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFkBplaysanimportantroleincontrollingmanyBIOLOGicalprocessesincludingimmuneandinflammatoryresponses,developmentalprocesses,cellulargrowth,andapoptosis.Inresponsetothevariousstimuli,suchasstress,cytokines,freerADIcals,ultravioletirradiation,andbacterialorviralantigens,NFkBisactivatedandtranslocatesfromcytoplasmtonucleus,whereNFkBbindstoitsresponseelementonthepromoterregionandregulatesawidespectrumofgeneexpression.DysfunctionofNFkBactivityisassociatedwithcancer,inflammatoryandautoimmunedisease,andviralinfection.MonitoringtheNFkBactivityisessentialtounveilthemechanismofthesediseasesandconductdrugdiscovery.
SignosishasestablishedaNFkBluciferasereporterstablecelllinethathasbeenstablytransfectedwithpTA-NFkB-luciferasereportervectorandcanbeusedforstudyingNFkBsignalingpathwaysactivatedbydifferentcytokines,suchasTNFαandmanyotherstimuli,enforcedgeneexpressionandgeneknockdown.ThecelllinewasestablishedbytransfectionusingapTA-NFkB-luciferasereportervector,whichcontains4repeatsofNFkBbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwithhygromycinexpressionvectorfollowedbyhygromycinselection.ThehygromycinresistantclonesweresubsequentlyscreenedforTNFa-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0013NFκBreporteractivityinresponsetoTNFαtreatment. TheMCF-7cellswereseededona96-wellplateforovernightincompletegrowthmedia. Thecellsthenweretreatedwithorwithout20ng/mlTNFαingrowthmediawith0.1%FBSfor16hours. MCF-7-NFkBLuciferaseReporterCellLineexhibitsTNFα-dependentincreaseinluciferaseactivitywhencomparedtountreatedcells.
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推荐了解“主细胞库”“工作细胞库”这两个名词。
① 在构建载体时，目的基因直接整合到细胞染色体组上，最好不要通过先瞬转在筛选稳定细胞株的这种方法，因为转染效率没有保证
② 高表达载体的构建，哺乳动物表达量一直是它自身的缺点，最好根据高表达载体定向的驯化细胞，提高蛋白表达量
③ 细胞的选择，筛选稳定细胞株我们常用的细胞是CHO，中国仓鼠卵巢细胞，由于CHO具有诸多的优点因此适合用于筛选稳定细胞株，而HEK293细胞则常用于瞬时转染
④ 后期的筛选，双抗预防污染，筛选细胞的时候抗生素浓度一定要做预实验，而且转染的时候不能有抗生素，关于细胞转染 稳定细胞系构建的相关理论
先提取RNA,反转录成cDNA,然后根据目的基因设计PCR引物,通过半定量RT-PCR确定目的基因表达.
正因为检测的是mRNA,所以要先反转录成cDNA才能PCR.
建立稳定细胞株,一般是根据不同基因载体中所含有的抗性标志选用相应的药物对靶...专注整体实验·服务生命科学已开展了数千个实验外包项目
