APAssayandFGFBinding

MikeNaski

COS-7cellsweretransfectedbyastandardDEAE-dextranmethodwith4mgofplasmid/106cells.Conditionedmediumwasharvested3,6and9daysaftertransfectionandusedforAPenzymeactivityandtodeterminetherelativeconcentrationofthesolublereceptors.Bindingofthereceptortoanimmobilizedheparin-FGFcomplexwasdoneaspreviouslydescribed(Grayetal.,1996).Briefly,10µlbedvolumeheparin-sepharosewasmixed1:1withsepharoseCL6Band100ngFGF2,FGF4orFGF8.AfteraddingPBSto50ml,themixtureswererotatedatroomtemperaturefor1hour,thenwashedtwicewith100µlPBSbeforetheadditionofreceptorsupernatant(800mOD/minute).Followingtheadditionofreceptorsupernatant,themixtureswerefurtherincubatedatroomtemperaturefor4hours,thenwashedthreetimeswith200µl20mMHepespH7.5,150mMNaCl,1%TritonX-100,10%glycerol,andtwicewith150µl10mMTrispH7.5,0.5MNaCl.Thereceptor-FGF-heparinsepharosecomplexwastransferredin60µl10mMTrispH7.5,toawellofamicrotiterplatecontaining60µlof2«APassaybuffer(2Mdiethanolamine,1mMMgCl2,20mMhomoarginine,12mMp-nitrophenylphosphatepH9.5).Theboundreceptorwasquantifiedbymeasuringtheinitialrateofsubstratehydrolysisat405nmandsameamountofproteinswasusedinthebindingassays.

Gray,T.E.,Eisenstein,M.,Yayon,A.andGivol,D.(1996).Asparagine-344IsaKeyResidueForLigandBindingInKeratinocyteGrowthFactorReceptor.Biochemistry35,15640-15645.

PCRAssayforFGFR3-/-Matings

Oligos...5"oligo(JC14):5"-GGGCTCCTTATTGGACTCGC-3"3"oligowild-typeallele(JC41):5"-AGGTATAGTTGCCACGATCGGAGGG-3"3"oligoknockoutallele(JC18):5"-TGCTAAAGCGCATGCTCCAGACTGC-3"

Fragmentlengths...wild-typefragment(JC14/JC41):322bpknockoutfragment(JC14/JC18):221bp

PCRConditions...BoehringerMannheimTaqPolymeraseand10XPCRbuffer.PCRprogram:95¡CX3";30X(95¡CX1",55¡CX1",72¡CX3");72¡CX3"

PCRforFGF9KOmiceOligos...5’oligo:(JC46):5’-GCAAGGGAGGGGAGTTGGATATACC-3’3’oligo:(JC42):5’-CAGCCCAAGCTTTCGCGAGCTCG-3’(inpolII-neo)3’oligo:(JC48):5’-GAAATCCAGTCCTGCAGTACAGCTGC-3’PCRConditions...BoehringerMannheimTaqPolymeraseand10XBMPCRbufferwithDMSO.PCRprogram:95Cx3’;30x(95Cx1’55Cx1’72Cx3’);72Cx3’ProductSize...wildtype:310bpTargettedallele:185bp

PCRforFGF17KOmiceNoPCRavailableforF17kogenotyping.

PCRassayforhGH(colII-FGFR3mice)Oligos...5’oligo:5’AGGTGGCCTTTGACACCTACCAGG3’3’oligo:5’TCTGTTGTGTTTCCTCCCTGTTGG3’Amplify360bpofhGHsequencepresentin3’UTRofthetransgene.PCRamplificationconditions...27cyclesof94°Cx60seconds,55°Cx60secondsand72°Cx90seconds.