
Description:
Hypoxia-InducIBLeFactor(HIF)-1isadimericproteincomplexthatplaysacentralroleintheresponsetolowoxygenconcentrations,orhypoxia,and isacrucialphysiologicalregulatorofhomeostasis,vascularization,andanaerobicmetabolism.HIFisatranscriptionfactorregulatinggeneexpressionbybindingtotheirDNArecognitionsiteonthetargetgenes.FurThermore,HIF-1hasbeenwidelystudiedbecauseofitsperceivedtherapeuticpotential. HIF-1allowssurvivalandproliferationofcancerouscellsduetoitsangiogenicproperties,andtheinhibitionpotentiallycouldpreventthespreadofcancer.WithagrowingunderstandingoftheHIF-1pathway,ithasbecomeanattractivegoaltoanalyzetheinhibitionandstimulationofHIFtranscriptionalactivityviasmallmolecules.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. |
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至于到底好不好转就跟你的细胞有关了,和细胞是否是稳转株关系不大。
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。
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。
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。

