Description:
NRF2/AREResponsiveLuciferaseReporterMCF7StableCellLineisderivedfromhumanbreastcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNRF2/AREresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofAntioxidantReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NRF2isatranscriptionfactor,whichplaysanimportantroleinrespondingtooxidativestress.Undernormalcellularconditions,NRF2formsaproteincomplexwithKeap1inthecytoplasm,whichresultsintheproteasomaldegradationofNRF2causingittobeinactive. OxidativestressleadstotheactivationofanumberofkinasesincludingMAPK,ERK,p38,PKC,andPI3K. TheyphosphorylatebothKeap1andNRF2,whichdisrupttheKeap1-NRF2complex,andstimulatethetranslocationofNRF2tothenucleus,whereitformsacomplexwithMafproteins. TheNRF2/Mafheterodimersbinddirectlytoantioxidantresponseelements(AREs)locatedwithinpromotersofNRF2targetgenesandcoordinatetheexpressionofantioxidantgeneproducts.
SignosishasdevelopedaNRF2-reporterMCF7stablecelllinethathasbeen stablytransfectedwithpTA-NRF2-luciferasereportervector,whichcontains8repeatsofNRF2bindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwithaG418expressionvector. Thiscelllinecanbeusedtoinvestigateoxidativestress-mediatedactivationofupstreamkinasesandtoscreenanticancerdrugsthatcaninduceARE-drivengeneexpression.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisoftheNRF2PathwayReporterMCF7StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout50nMTBHQrespectivelyinDMEMand0.1%FBSfor16hours.
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由于质粒的不兼容性,拥有同种复制子的质粒不能在同一细胞内稳定共存,经过几代的复制,会质粒丢失,所以并不是任何两个或两个以上质粒都可以在同一细胞内稳定存在,但是可以同时进入.
稳定转染的细胞株,就是转染后质粒可以稳定整合到基因组上不会因细胞分裂而丢失。区别于质粒瞬时转染不能长时间保留质粒在细胞里。
如果经费充足的话可以找公司包装病毒,如武汉的普健可以提供各种载体的构建及细胞株构建的技术服务。
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.
2.将构建完成的载体与慢病毒包装质粒混合,共转染靶细胞
3.收集病毒液
4.用病毒液感染靶细胞
5.用载体上带的抗生素进行筛选,如果没有,可以用无限稀释法
6.获得稳转株