title: An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.
abstract: Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.
authors.affiliation: Department of Biology, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Marie S
authors.initials: MS
authors.lastname: Isidor
authors.affiliation: Department of Biology, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Sally
authors.initials: S
authors.lastname: Winther
authors.affiliation: Department of Biology, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Astrid L
authors.initials: AL
authors.lastname: Basse
authors.affiliation: Department of Biology, University of Copenhagen , Copenhagen, Denmark.
authors.forename: M Christine H
authors.initials: MC
authors.lastname: Petersen
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University , Stockholm, Sweden.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cannon
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University , Stockholm, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Nedergaard
authors.affiliation: Department of Biology, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Jacob B
authors.initials: JB
authors.lastname: Hansen
keywords: Seahorse; Ucp1; brown adipocytes; human adipocytes; insulin signaling; knockdown; lipolysis; primary adipocytes; reverse transfection; siRNA