Description:

Principle:

Nuclearhormonereceptors(NHRs)areagroupofligand-bindingtranscriptionfactors(TFs). Morethan350NHRsareavailableinthePDB.LikeotherTFs,theyareabletoregulategeneexpressionbybindingtospecificDNAregulatoryelements,buttheiractivitiesaremodulatedbythecorrespondingligands.Theyplaymanyimportantphysiologicalfunctionssuchasembryonicdevelopment,organphysiology,celldifferentiationandhomeostasis.Theyarealsoassociatedwithhumandiseasesincludingcancer,obesity/diabetes,arthritisandasthma.Membersofthisfamilyareoftenchosentobedrugtargets.

Conventionally,theresponseelement-luciferasereporterassaysareusedtostudyendogenousorexogenousreceptors,andanalyzethereceptorsignalingpathwayinanativeBIOLOGicalcontext. DuetotheirlowsensitivityoftheseassaysandthesimilarresponseelementsharedbymultipleNHRs,theirapplicationsarelimited. SignosishasdevelopedLDB-drivenGAL4reporterHEK293stablecelllinesbydeliveringtwovectors,oneexpressing8copiesofGAL4upstreamactivatorsequences(UAS)infrontoftheluciferasereportergene,anotherexpressingGal4DNABindingDomain(DBD)andGRligandbindingdomain(LBD).Uponadditionofacorrespondingligand,theDBD-LBDfusionproteinisactivated,andbindstoGALpromoterbindingsite,leADIngtotheinductionofluciferase. ThestablecloneswereselectedwithhygromycinandG418,ThefunctionalassaywassubsequentlyconductedbyDEXtreatment,andtheclonewiththehighestinductionfold(50)hasbeenchosenforthisproduct.Theadvantagesofthesestablecelllinesarelowcross-reactivitywithothernuclearreceptorsandlesstoxicityofthechimericreceptorstothecellswhenoverexpressed. ThesecelllinescanbeusedtoscreendrugcompoundsforagoNISTorantagonisthitidentification.