Biology Reference
In-Depth Information
function of LDs at the hub of cellular energy homeostasis regulation, major questions
in the field of LD biology are still unanswered.
Drosophila melanogaster
has been
used as a model organism to make fundamental discoveries in biology for over a cen-
tury. In recent years, genome-wide unbiased reverse genetic screens using
Drosoph-
ila
cells or transgenic lines have been proven to provide valuable knowledge to the
field of LD biology. Here we summarize the methods we use for functional genomic
screens in
Drosophila
S2 cells to identify genes involved in LD biology, and the
methods used for studying LD function
in vivo
using
Drosophila
as a model to com-
bat metabolic diseases.
4.1
GENOME-WIDE RNA INTERFERENCE SCREENS
IN DROSOPHILA CELLS TO IDENTIFY REGULATORS FOR
LD BIOLOGY
Three
Drosophila
genome-wide RNAi screens have been performed in recent years
aimed at discovering regulators for lipid droplet (LD) biology in cells (
Beller et al.,
2008; Guo et al., 2008
) and fat storage in whole organisms (
Pospisilik et al., 2010
).
Of the three screens, two were performed in
Drosophila
culture cells (S2 cells in Guo
et al., Kc
167
cells in Beller et al.), and one used transgenic RNAi lines (
Pospisilik
et al., 2010
). Here we describe the design, rationale, screening procedure, and results
of our S2 cell screen.
4.1.1
S2 cells and their advantage for RNAi screens
Drosophila
Schneider 2 (S2) cells were derived from primary cultures of 20-24-h-
old
Drosophila
embryos more than 40 years ago (
Schneider, 1972
). It is the most
commonly used
Drosophila
cultured cell line and can be grown at room temperature
without the use of tissue-culture incubators. They grow both in suspension and as a
loose monolayer and thus can be lifted by gentle pipetting.
Drosophila
S2 cells con-
tain numerous small LDs under basal culture conditions and rapidly form LD clusters
upon fatty acid supplement in the medium (
Guo et al., 2008
). Many human lipid me-
tabolism genes are conserved in flies (
Baker & Thummel, 2007; Kuhnlein, 2012
).
These features make S2 cells a good model to study LD biology
in vitro
. S2 cells
robustly uptake double-stranded RNAs (dsRNA) from culture medium through scav-
enger receptor-mediated endocytosis (
Ulvila et al., 2006
). The endocytosed dsRNAs
induce efficient RNA interference responses in S2 cells. The targeted genes are
downregulated 80-90% on average (
Goshima et al., 2007; Guo et al., 2008
). These
features ensure efficient RNAi and greatly reduce the cost of performing genome-
wide RNAi screens compared to using mammalian cells, which require costly
methods to introduce siRNA or shRNA into the cells and is difficult to achieve a high
knockdown efficiency.
