Studying lipolysis in adipocytes by combining siRNA knockdown and adenovirus-mediated overexpression approaches

Xiaodong Zhang, Bradlee L. Heckmann, Jun Liu

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

3T3-L1 adipocytes are widely used as a model system for studying hormone-stimulated lipolysis. However, these cells were limited in their utility for gain- and loss-of-function studies due to the low efficiency of their transfection with plasmid DNA or small interfering RNA (siRNA) oligos. In this chapter, we provide a review of two methods established for manipulation of protein expression in differentiated mature adipocytes. The use of electroporation allows a high-efficiency delivery of siRNA oligos and subsequent knockdown of specific gene expression. A centrifugation-assisted infection with recombinant adenovirus, on the other hand, enables robust overexpression of ectopic proteins. Most importantly, by combining siRNA electroporation with adenovirus infection, simultaneous manipulation of levels of two different proteins can be achieved in differentiated adipocytes. Through subsequent analyses of lipase activity in cell extracts and fatty acid or glycerol release from living cells, mutual interdependence between the two proteins in the context of basal and hormone-stimulated adipocyte lipolysis can be evaluated.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc
Pages83-105
Number of pages23
DOIs
StatePublished - 2013

Publication series

NameMethods in Cell Biology
Volume116
ISSN (Print)0091-679X

Keywords

  • Adenovirus
  • Adipocytes
  • ATGL
  • Fatty acid
  • Knockdown
  • Lipid droplet
  • Lipolysis
  • Overexpression
  • SiRNA
  • Triacylglycerol

ASJC Scopus subject areas

  • Cell Biology

Fingerprint Dive into the research topics of 'Studying lipolysis in adipocytes by combining siRNA knockdown and adenovirus-mediated overexpression approaches'. Together they form a unique fingerprint.

  • Cite this

    Zhang, X., Heckmann, B. L., & Liu, J. (2013). Studying lipolysis in adipocytes by combining siRNA knockdown and adenovirus-mediated overexpression approaches. In Methods in Cell Biology (pp. 83-105). (Methods in Cell Biology; Vol. 116). Academic Press Inc. https://doi.org/10.1016/B978-0-12-408051-5.00006-1