New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.

Since its introduction as a vital marker for gene expression in the mid-1990s (Chalfie et al., 1994), the Aequorea victoria green fluorescent protein (GFP) and its derivatives have become an invaluable research tool to investigators working in a multitude of experimental systems. The molecule’s small size (238 amino acids) and stability enable it to function when fused to a wide variety of proteins and protein localization domains, allowing for nuclear, membrane-bound, cytoskeletal, and organelle-specific vital imaging at a number of wavelengths and with emission in several visually separable spectra (reviewed by De Giorgi et al., 1999). The potential for fluorescence resonance energy transfer (FRET) between different GFP variants, such as the cyan (CFP) and yellow (YFP) fluorescent proteins, additionally allows the use of these markers to detect protein–protein interactions within living cells (Pollok and Heim, 1999). One of the fluorescent proteins’ most useful functions, however, remains that of a simple vital reporter for gene expression and a marker for discrete cell types within the living organism. Although the fluorescent proteins have been used with good success in Drosophila, their performance has sometimes been disappointing when used in attempts to visualize gene expression in the developing Drosophila embryo (e.g., Davis et al., 1995). This is largely attributable to two factors: the time-lag between expression of GFP and its acquisition of fluorescence (Heim et al., 1994), and the autofluorescence of the yolk. The latter problem can be partially overcome by judicious selection of barrier filters or by confocal microscopy, while the former has been mitigated to a degree by the development of “enhanced” fluorescent protein derivatives that are both brighter and quicker to acquire fluorescence than the wild-type GFP (Cormack et al., 1996; Heim et al., 1994, 1995; Heim and Tsien, 1996). Nevertheless, detection of fluorescence can still be difficult in the rapidly developing embryo. We describe here a set of dicistronic GFP and YFP reporters constructed for use with the GAL4/UAS expression system (Brand and Perrimon, 1993), which, as a result of their containing two copies of the respective fluorescent protein gene, are significantly brighter and thus more easily and rapidly detectable than currently available UAS-GFP fly stocks (Dickson, 1996; Yeh et al., 1995). We also describe a set of GFP-marked balancer chromosomes with robust embryonic expression that is detectable at an earlier timepoint than existing GFP balancers (Casso et al., 2000). Transgenic flies were created in which two copies of the “enhanced” fluorescent protein derivatives EGFP or EYFP were separated by the internal ribosome entry site (IRES) from the Drosophila Ubx gene (Hart and Bienz, 1996) and regulated by means of the GAL4/UAS expression system (Fig. 1). Lines on each chromosome that exhibited bright fluorescence when crossed to Gal4containing flies were chosen as the UAS-2xEGFP and UAS-2xEYFP stocks (Table 1) and tested in a variety of settings by examining fluorescence in embryos, egg chambers, larvae, and imaginal discs (Fig. 2a–h, and data not shown). Fluorescence intensity was significantly brighter than that seen when the identical Gal4 drivers were crossed to flies containing the UAS-GFP[S65T] reporter construct (Dickson, 1996; data not shown). This increase in intensity is partially due to the brighter nature of the EGFP derivative compared to the S65T derivative (Cormack et al., 1996) and partially due to the presence of the extra copy of EGFP in the dicistronic construct. Flow cytometric analysis confirms that the 2xEGFP construct is roughly two-fold brighter than a single-copy EGFP construct when expressed in S2 cells (Halfon, 1998). The UAS-2xEYFP flies should be useful in conjunction with CFP for FRET-based experiments and should also be particularly well-suited for imaging embryonic structures that lie near to the autofluorescent gut, as EYFP can be detected under conditions where autofluorescence is minimal (e.g., Leica YFP filter set,