Prolonged efficiency of siRNA-mediated gene silencing in primary cultures of human preadipocytes and adipocytes.

Abstract

Primary human preadipocytes and differentiated adipocytes in culture are valuable cell culture systems to study adipogenesis and adipose function in relation to human adipose biology. To use these systems for mechanistic studies, siRNA-mediated knockdown of genes for its effectiveness was studied. Methods were developed to effectively deliver siRNA for gene silencing in primary preadipocytes isolated from human subcutaneous adipose tissue and newly differentiated adipocytes. Expression level of genes and proteins was measured using quantitative RT-PCR and western blotting. Lipid droplet morphology was observed using microscopy, and glycerol release was quantified as a measure of lipolysis. siRNA-mediated knockdown of genes in primary human preadipocytes resulted in prolonged silencing effects, suppressing genes throughout the process of their differentiation. In newly differentiated adipocytes, siRNA-mediated gene knockdown allowed proteins to stay depleted for at least 5 days. It was possible to re-express a protein after its siRNA-mediated depletion. Importantly, siRNA transfected human adipocytes remained metabolically active, responding to β-adrenergic stimulation to increase lipolysis. Our study describes the methods of gene silencing in primary cultures of human preadipocytes and adipocytes and their prolonged effectiveness.