Figure 1

Enhancement of protein expression by refinement of UAS vector. (A) Structure of UAS vectors used for GFP expression in the present study. Upper panel indicates conventional UAS vector used to generate UAS-mCD8GFP strain previously. Lower panel indicates improved vector for UAS-myrGFP strain, which has increased copies of the GAL4 binding sites (20xUAS), an intervening sequence derived from myosin heavy chain gene of Drosophila melanogaster (IVS), synthetic translational enhancer (Syn21), and 3′UTR of AcNPV p10 gene (p10T). mCD8 (mouse CD8) and myr (N-myristoylation) sequences are membrane targeting signals. (B) Relative expression levels of GFP mRNA in the male antennae determined by qRT-PCR. UAS-mCD8GFP and UAS-myrGFP strains were crossed to BmOR1-GAL4 driver, which expresses GAL4 in the major sex pheromone receptor (BmOR1)-expressing cells. (C) Bright field and fluorescent images of dissected male brains under an epifluorescent microscope. Bright GFP signal was observed in the antennal nerves and antennal lobes of the myrGFP-expressed moth, whereas GFP fluorescence was barely observed in those of the mCD8GFP-expressed moth. (D) Quantification of GFP fluorescence intensities in the antennal lobes. (E) Confocal images of immunostained antennal lobes. Images of upper panels were taken with the same laser power. GFP signal could be observed with a higher laser power in the mCD8GFP-expressed sample (lower panel). (F) Relative fluorescent intensities of GFP in toroid, where BmOR1-expressing cells input. (G) Western blot analyses of GFP and α-tubulin of the brains (BR) and antennae (AN). Expected sizes of mCD8GFP and myrGFP are 51 and 36 kDa, respectively. Under the same exposure time, signal was detected only in the myrGFP-expressed samples. *P < 0.0001, **P < 0.00001, t-test. Number of samples are indicated in the parentheses. Bars, 500 μm (C) and 100 μm (E).