Plightswitch hucp1 prom

Immortalized human fat progenitor cells were infected with a lentivirus containing the plasmid, pLV.ExBi.P/Puro-hUCP1promoter-Luc(firefly)-T2A-hrGFP that expresses luciferase and GFP driven by human UCP1 promoter. 4148bp human UCP1 promoter was cloned from pLightSwitch_hUCP1-Prom (S723122; Switch Gear Genomics, Carlsbad, CA) and was then sub-cloned into a lentiviral plasmid to generate plasmid containing a UCP1 reporter system (Cyagen Biosciences Inc., Santa Clara, CA). 293T cells (ATCC) were transfected with hUCP1promoter-Luc-T2A-GFP, pMD2.G and psPAX2 DNA using PolyJet DNA in vitro transfection reagent (SignaGen Laboratories, Rockville, MD). Culture supernatants containing virus were collected every 24 h after infection and filtered through a 0.45 μm filter (Fisher, Scientific, Pittsburgh, PA). Immortalized human white and brown fat progenitors at 80% confluence were infected with viral supernatants in the presence of 4 μg/mL Polybrene every day until cells reached 90% confluence. Then cells were treated with 1 μg/mL puromycin in DMEM/H medium containing 10% FBS and antibiotics. Once drug selection was finished, the cells were maintained in culture medium with 0.2 μg/mL puromycin for 2 weeks.