Description:
p53 ResponsiveLuciferaseReporterRKOStableCellLineisderivedfromhumancoloncancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofthep53responseelement.Thiscelllineisanidealcellularmodelformonitoringtheactivationofp53Pathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Thep53pathwayplaysacrucialroleineffectivetumorsuppressionbecauseofitscentralfunctionincellcycleregulation,DNArepair,cellularsenescence,andapoptosis,whichcanbeusedforpotentiallydevelopnewdrugtherapiesagainstcancer.UponactivationbyDNAdamage,oncogeneactivation,orhypoxia,p53bindstoitsDNArecognitionsiteonthepromoterregionsofthetargetgenesandregulatethegeneexpression. Signosishasestablishedp53luciferasereporterstablecellline,inwhichluciferaseactivityisspecificallyassociatedwiththeactivityofp53.Therefore,thecelllinecanbeusedasareportersystemformonitoringtheactivationofp53triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.
Thecelllinewasestablishedbytransfectionofp53luciferasereportervectoralongwithG418expressionvectorfollowedbyG418selection.TheG418resistantclonesweresubsequentlyscreenedforetoposide-inducedluciferaseactivity.Theclonewiththehighestfoldinduction(35fold)wasselectedandexpandedtoproducethisstablecellline.

PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:

Analysisofp53PathwayReporterRKO StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout2ug/mlQuinacrinerespectivelyinDMEMand0.1%FBSfor16hours.
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推荐了解“主细胞库”“工作细胞库”这两个名词。
1.将外源基因插入慢病毒载体
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
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。

