Description:
CREBResponsiveLuciferaseReporterHEK293StableCellLineisderivedfromhumanembryonickidney,andstablyexpressfireflyluciferasereportergeneunderthecontrolofCREBresponseelement.ThiscelllineisanidealcellularmodelformonitoringtheactivationofcAMP,PKA,CaMKSignaling ReceptorSignalingPathwaytriggeredbystimulitreatment,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-0020CREBreporteractivityinresponsetoforskolintreatment. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10uMforkoslininDMEMand0.1%FBSfor16hours. Forskolininducedover50-foldincreaseinluciferaseactivitycomparedtountreatedcells.
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然后你得确定，这个细胞株对于你得研究来说不可或缺，具有明确的代表性。
上面两个都没有问题的话，祝你顺利。
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
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。
