
Description:
p53ResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer ,andstablyexpressfireflyluciferasereportergeneunderthecontrolofthep53responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivationofp53ReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Thep53pathwayplaysacrucialroleineffectivetumorsuppressionbecauseofitscentralfunctionincellcycleregulation,DNArepair,cellularsenescence,andapoptosis,whichcanbeusedforpotentiallydevelopnewdrugtherapiesagainstcancer.UponactivationbyDNAdamage,oncogeneactivation,orhypoxia,p53bindstoitsDNArecognitionsiteonthepromoterregionsofthetargetgenesandregulatethegeneexpression. Signosishasdevelopedp53luciferasereporterHelastablecellline,inwhichluciferaseactivityisspecificallyassociatedwiththeactivityofp53.Therefore,thecelllinecanbeusedasareportersystemformonitoringtheactivationofp53triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.
Thecelllinewasestablishedbytransfection usinga pTA-p53-luciferasereportervector,whichcontains6repeatsofp53bindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. Thehygromycinresistantclonesweresubsequentlyscreenedfordoxorubicin-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
Analysisofp53ReporterHelaStableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenwereeithertreatedwith500ng/mlDoxorubicinor2ug/mlQuinacrinerespectivelyinDMEMand10%FBSfor16hours.
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推荐了解“主细胞库”“工作细胞库”这两个名词。
推荐了解“主细胞库”“工作细胞库”这两个名词。
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.
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。

