New series of Drosophila expression vectors suitable for behavioral rescue.

Drosophila melanogaster remains an excellent model system for understanding complex biological processes. This is in large part due to the many tools that can be applied experimentally. The control of transgene expression in Drosophila has been an extremely successful technique for assessing genetic function. P-element mediated germ-line transformation is routinely used to generate novel Drosophila genotypes for gain-of-function studies. Behavior is an extremely complex process that might rely on subtle changes in cellular physiology and gene expression. Nevertheless, behavior is amenable to examination through the ectopic expression of target genes; however, care must be taken to use Pelement vectors that are neutral. We have constructed new Drosophila expression vectors that use either the heat-shock protein 70 (Hsp70Bb) or the synthetic Gal4/UAS promoters with the neutral rosy (ry) selectable marker. They are thus suitable for behavioral gain-of-function studies (Figure 1). The majority of currently used P-element vectors utilize a genetically engineered white gene (mini-w) as a selectable marker (7,8). The mini-w marker has several useful features, including an easily scoreable, semidominant eye pigmentation phenotype. However, this marker has been found to induce dramatic behavioral changes when ectopically expressed (5,10). Specifically, induced mini-w expression leads to robust male-male courtship. The mechanism responsible for this behavioral change is unknown, and it may confound experiments designed to examine other behaviors or aspects of central nervous system (CNS) function. Therefore, we constructed three P-element expression vectors that utilize the ry gene as a selectable marker. Similar to mini-w, ry has an easily scoreable dominant eye pigmentation phenotype. Additionally, many identified Drosophila learning and memory mutants have been isolated in a rybackground (4), making ry a very useful marker for phenotypic rescue in these lines. Lastly, rymarked transposons have been found to be neutral in olfactory associative learning (3,9). Our first two vectors utilize the Hsp70Bb promoter to achieve temporal control of gene expression. We call these vectors pPBretH/XH or HX for Baylor College of Medicine, ry expression transposon, Hsp70 promoter. The XH or HX denote the orientation of the multiple cloning site (MCS). Basal-level expression from this promoter is extremely low at room temperature; however, it can be induced as much as 1000-fold by heat shock (2). Our third vector uses the heterologous Gal4/UAS promoter to achieve tissue-specific expression through the Gal4 binary system (1). We named this vector pBretU, where U represents the upstream activator sequence (UAS) promoter. This UAS promoter contains five optimized Gal4-binding sites followed by an Hsp70 TATA box (1). Requisite for this promoter is a Gal4-driver transposon. A large number of transposon are availBenchmarks