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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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。
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。
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。
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
