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小鼠脂肪组织特异表达faT-1基因载体的构建.docx

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小鼠脂肪组织特异表达faT-1基因载体的构建 Introduction Obesityisaglobalhealthissuethataffectsmillionsofpeopleworldwide.Increasedadiposityhasbeenassociatedwithanincreasedriskofmetabolicdisorders,suchasdiabetesandcardiovasculardiseases.Therefore,therehasbeenagrowinginterestinstudyingthemolecularmechanismsthatregulateadiposityandidentifyingnewmoleculartargetsfortherapeuticinterventions. Fattyacidsareoneofthemajorcomponentsofadiposetissueandplayacrucialroleinregulatingadiposity.Fattyacidtransportproteins(FATPs)areagroupofmembraneproteinsthatareinvolvedinfattyaciduptakeandmetabolisminvarioustissues,includingadiposetissue.FATP1isoneofthemajorisoformsofFATPsandhasbeenshowntobeexpressedinadiposetissue.StudieshaveshownthatFATP1playsanimportantroleinregulatingadiposetissuemetabolismandadiposity.Therefore,inthisstudy,weaimedtoconstructafaT-1geneexpressionvectorspecifictomouseadiposetissuetoelucidatethemolecularmechanismsunderlyingadiposityregulation. MaterialsandMethods ConstructionofthefaT-1geneexpressionvector ThefaT-1genewassynthesizedbyGenScriptCorporation(Nanjing,China).ThesequenceoffaT-1wasoptimizedforexpressioninmouseadiposetissueandwasclonedintoapLenti-CMV-purovector(Addgene,MA,USA)usingstandardmolecularbiologytechniques.Theresultingexpressionvector,pLenti-CMV-puro-faT-1,wasverifiedbysequencing. Cellcultureandtransfection 3T3-L1cells(ATCC,VA,USA)wereculturedinDMEM(Dulbecco'smodifiedEagle'smedium)supplementedwith10%fetalbovineserum(FBS)and1%penicillin-streptomycin(PS)inahumidifiedincubatorwith5%CO2at37°C.ThecellsweretransfectedwiththepLenti-CMV-puro-faT-1ortheemptypLenti-CMV-purovectorusingLipofectamine3000(ThermoFisherScientific,MA,USA)accordingtothemanufacturer'sinstructions.Thetransfectedcellswereselectedwith2μg/mlpuromycinfor48hourstoestablishstablecelllinesexpressingfaT-1oranemptyvectorcontrol. Real-timePCR TotalRNAwasextractedfromthe3T3-L1cellsusingTRIzolreagent(ThermoFisherScientific,MA,USA)accordingtothemanufacturer'sinstructions.cDNAwassynthesizedfrom1μgoftotalRNAusingtheSuperScr