Description:
HIFResponsiveLuciferaseReporterNIH/3T3StableCellLineisderivedfrommousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheHIFresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofHypoxiaReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Hypoxia-InducIBLeFactor(HIF)-1isadimericproteincomplexthatplaysacentralroleintheresponsetolowoxygenconcentrations,orhypoxia,and isacrucialphysiologicalregulatorofhomeostasis,vascularization,andanaerobicmetabolism.HIFisatranscriptionfactorregulatinggeneexpressionbybindingtotheirDNArecognitionsiteonthetargetgenes.FurThermore,HIF-1hasbeenwidelystudiedbecauseofitsperceivedtherapeuticpotential. HIF-1allowssurvivalandproliferationofcancerouscellsduetoitsangiogenicproperties,andtheinhibitionpotentiallycouldpreventthespreadofcancer.WithagrowingunderstandingoftheHIF-1pathway,ithasbecomeanattractivegoaltoanalyzetheinhibitionandstimulationofHIFtranscriptionalactivityviasmallmolecules.
Thecelllinewasestablishedbytransfection usinga pTA-HIF-luciferasereportervector,whichcontains4repeatsofHIFbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforCoCl2-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
HIFNIH3T3StableCellLineAnalysis. HIFNIH3T3stablecellswereseededina96-wellcultureplatewiththeindicatedmedium. Thecellsweretreatedwith100uMCoCl2for8hours.
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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。
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.
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
