Description:
NFAT ResponsiveLuciferaseReporterJurkatT StableCellLineisderivedfromhumanTlymphocyte,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFATresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofCalciumSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFATsareafamilyoftranscriptionalfactorsthatplayanimportantroleinimmuneresponseaswellasinthedevelopmentofcardiac,skeletalmuscle,andnervoussystems. NFATsareregulatedbycalciumsignaling.Throughcalciumactivationofthephosphatasecalcineurin,NFATcproteinstranslocatefromthecytoplasmintothenucleus,wheretheycooperatewithotherproteinstomediategeneexpression.NuclearimportofNFATisblockedbykinasessuchasPKAandGSK3.NFATsarealsoimplicatedinbreastcancer. SignosishasestablishedaNFATluciferasereportercelllinethathasbeenstablytransfectedwithaNFAT-luciferasereporterconstruct.Viatheanalysisofluciferase,thecelllinecanbeemployedtomonitorthecellularchangesofNFATactivitiesthataretriggeredbystimulation,compoundtreatment,enforcedgeneexpressionorgeneknockdown.
Thecelllineisestablishedbytransfectionusinga pTA-NFAT-luciferasereportervector,whichcontainsNFATbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforPMA+ionomycin-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofNFATLuciferaseReporterJurkatTStableCellLine.Thecellswereseededon a96-wellplateinmediacontaining10ng/mlPMA,1uMionomycin,and0.1%FBSfor16hours. Morethan200foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.
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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
对于瞬时转染的检测:如果有报告基因,就通过报告基因的表达看转染成功与否;没有报告基因的话,就在24~96h内收获细胞通过RT-PCR和WB来鉴定。
推荐了解“主细胞库”“工作细胞库”这两个名词。
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
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。
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