Biology Reference
In-Depth Information
lipolysis (
Liu, Zhou, Abumrad, & Su, 2010; Snyder, Esselstyn, Loughney, Wolda, &
Florio, 2005; Yang et al., 2010
). To make them a feasible model for functional studies
on lipolytic regulators, however, it is essential to develop effective methods for manip-
ulation of gene expression in these cells. In this regard, the well-recognized difficulty in
introducing DNA or siRNA-targeting genes of interest into mature 3T3-L1 adipocytes
has presented a major technical challenge (
Ross, Song, Burney, Kasai, & Orlicky,
2003
). For ectopic gene expression, some investigators used electroporation method
to deliver plasmid DNA in differentiated cells (
Jiang, Chawla, Bose, Way, & Czech,
2002; Martin, Lee, & McGraw, 2006; Okada, Mori, & Pessin, 2003; Watson et al.,
2003
). While it produces a sufficient number of positive cells for imaging analysis,
the technique has limited usage due to the low transfection efficiency and thus is often
unreliable for assays that are dependent on examining a majority of the cell population.
Other investigators also attempted adenoviral gene transfer with varying degree of
success. To enhance the infection efficiency, Ross et al. have generated stable clones
of 3T3-L1 preadipocytes that exogenously express the coxsackie-adenovirus receptor
(
Ross et al., 2003
). However, how efficiently cells from those stable clones can be
differentiated into mature adipocytes with full lipolytic capacity remains unclear.
This chapter describes methods to effectively overexpress or knockdown specific
gene of interest in differentiated 3T3-L1 adipocytes. Knockdown is accomplished by
electroporation of cells in suspension with siRNA oligos. A centrifugation method is
employed to enhance the overexpression mediated by recombinant adenovirus.
Additionally, we describe the usage of these two methods in combination to achieve
manipulation of two different proteins simultaneously for the purpose of studying the
relevance of their interplay in the regulation of lipolysis.
6.1
MATERIALS AND REAGENTS
6.1.1
Equipment and materials
Gene Pulser Xcell
(Bio-Rad)
Electroporation cuvette (4-mm gap, Molecular Bioproducts, #5540)
Misonix sonicator (Qsonica, LLC, #XL-2000)
Scintillation counter (Beckman, #LS6500)
15- and 50-mL conical tubes (BD Falcon, #352097 and #352098)
Six-well and 15-cm tissue culture plate (vacuum gas plasma treated, BD Falcon,
#3506 and #353025)
2
™
75 mm glass test tube (VWR, #47729-570)
5-mL polypropylene round-bottom tubes (BD Falcon, #352063)
6.1.2
Reagents
Dulbecco's phosphate-buffered saline/modified (DPBS,
calcium-magnesium,
Thermo Scientific, # SH30028)
Dulbecco's modified Eagle's medium (DMEM with 4.5 g/L of glucose,
Cellgro
®
, #10-013-v)
