Description:
CREBResponsiveLuciferaseReporterNIH/3T3StableCellLineisderivedfrommousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontrolofCREBresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofcAMP,PKA,CaMKSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
CyclicAMPresponseelement(CRE)-bindingprotein(CREB)isatranscriptionfactorthatregulatesandrespondstodiversecellularresponses,rangingfromproliferation,survival,differentiation,stressresponses,andneuronalactivity. Thesecellularsignalsleadtoupstreamkinaseactivation,suchasproteinkinaseA(PKA),pp90ribosomalS6kinase(pp90RSK),andCa2+/calmodulin-dependentproteinkinases(CaMKs),andthesekinasesinturnphosphorylateCREBtoinduceCREBactivity. CREBincreasesthetranscriptionofgenesthatcontaincAMPresponsiveelements.
SignosishasdevelopedCREBluciferasereporterstablecelllinebyco-transfectingCREBluciferasereportervectorandhygromycinexpressionvector. Thehygromycinresistantclonesweresubsequentlyscreenedforforskolin-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivationofCREBtriggeredbystimulitreatment,suchasforskolinandgeneoverexpressionandgeneknockdown.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0031CREBreporteractivityinresponsetoforskolintreatment. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10uMforkoslininDMEMand0.1%FBSfor16hours. Forskolininducedover10-foldincreaseinluciferaseactivitycomparedtountreatedcells.
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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
一种是脂质体转染后,单克隆筛选稳定细胞株.另外一种是应用逆转录病毒,慢病毒转染,筛选稳定细胞株.
脂质体转染:在转染24小时后,消化细胞并计数.将细胞种到96孔板,保证每个孔2-3个细胞,这样才能得到单克隆.待细胞贴壁后,加入抗生素筛选.筛选时间和浓度视细胞而定.一般G418一个星期作用,嘌呤霉素2-3天.
脂质体法筛单克隆时间较长,且效率低,大概只有1%.
病毒转染:先要用包装细胞,一般为293细胞,包装出病毒,再用病毒转染目的细胞.包装病毒视不同类型的病毒而定,一般要3-5天的时间.包装好的病毒要测滴度,根据滴度决定转染目的细胞的病毒量.转染目的细胞1-2天后加抗生素筛选得到稳定细胞株.
病毒转染得到稳定细胞株的效率高,只是步骤繁琐.
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