Biology Reference
In-Depth Information
CHAPTER
6
Studying Lipolysis in
Adipocytes by Combining
siRNA Knockdown and
Adenovirus-Mediated
Overexpression Approaches
Xiaodong Zhang
*
,{
, Bradlee L. Heckmann
*
,{
, and Jun Liu
*
,{
*
Department of Biochemistry and Molecular Biology, Mayo Clinic in Arizona, Scottsdale,
Arizona, USA
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Metabolic HEALth Program, Mayo Clinic in Arizona, Scottsdale, Arizona, USA
CHAPTER OUTLINE
Introduction and Rationale......................................................................................... 84
6.1 Materials and Reagents...................................................................................... 87
6.1.1 Equipment and Materials .................................................................. 87
6.1.2 Reagents ......................................................................................... 87
6.1.3 Media and Stock Solutions ................................................................ 88
6.2 Methods ............................................................................................................ 89
6.2.1 Adipocyte Culture and Differentiation................................................. 89
6.2.2 Manipulation of Gene Expression ....................................................... 90
6.2.3 Functional Assays for Lipolysis .......................................................... 94
6.2.4 Case Study—evaluating the Roles of Perilipin 1 and CGI-58
in ATGL-mediated Lipolysis............................................................... 96
Concluding Remarks ................................................................................................. 97
Acknowledgment..................................................................................................... 100
References ............................................................................................................. 100
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
