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L
unc-119(+) vha-6 prom.
gfp
dgat-2 cDNA let-858 3'UTR
R
L
Mos1
R
Mos transposase mediated
transposon excision
L
unc-119(+) vha-6 prom.
gfp
dgat-2 cDNA let-858 3'UTR
R
L
R
homology arms guided recombination
transgene insertion
unc-119(+) vha-6 prom.
gfp
dgat-2 cDNA let-858 3'UTR
L
R
FIGURE 3.1
Generation of single copy transgenes. Mutant unc-119(
) animals carrying a single Mos1
transposon in the genome are used for single copy transgene insertion. The red boxes
depict the left (L) and right (R) homology arms that flank the Mos1 transposon. The same
homology arms flank the vha-6p::gfp::dgat-2::let-858 3'UTR transgene, placed next to a
genomic fragment of the C. briggsae unc-119(รพ) gene. Rescue of the unc-119() paralysis
phenotype indicates potential insertion events.
Adapted from
Frokjaer-Jensen et al. (2008)
.
enzyme for triglyceride synthesis and it is constitutively localized to the lipid droplet
surface in
C. elegans
(
Xu et al., 2012; Yen, Stone, Koliwad, Harris, & Farese, 2008
).
In mammalian cells, the DGAT-2 ortholog also associates with lipid droplets
during active triglyceride synthesis after lipid loading (
Kuerschner, Moessinger,
& Thiele, 2008; McFie, Banman, Kary, & Stone, 2011; Stone et al., 2009
). Since
the intestine is the major fat storage tissue in
C. elegans
, the transgenes were driven
by the intestinal specific
vha-6
promoter (
Oka, Toyomura, Honjo, Wada, & Futai,
2001
). Other intestinal specific promoters, such as those from the
elt-2
and
ges-1
genes, can also be used (
Aamodt, Chung, & McGhee, 1991; Fukushige, Hawkins,
& McGhee, 1998
). To ensure that the GFP::DGAT-2 and mRuby::DGAT-2 fusion
proteins were functional, we showed that they could support lipid droplet expansion
and compensate for the loss of endogenous DGAT-2 protein in
dgat-2(
)
mutant
animals (
Xu et al., 2012
). The repertoire of lipid droplet associated proteins in
C. elegans
has been greatly expanded recently through a comprehensive proteomic
study (
Zhang et al., 2012
). More than 300 proteins were identified and at least one of
them (DHS-3) is localized to lipid droplets when fused to GFP. We expect the num-
ber of lipid droplet markers for live imaging in
C. elegans
to increase exponentially
in the near future.
