Conditional expression of UAS‐transgenes in the adult eye with a new gene‐switch vector system

The spatial control of transgene expression with the Gal4 binary system has been one of the most productive experimental techniques applied to Drosophila melanogaster (Brand and Perrimon, 1993). However, the inability to temporally regulate the timing of gene expression can frequently make some experiments involving later developmental stages extremely difficult or even unachievable. Also, the lack of experimenter’s control over the induction of gene expression usually makes withingenotype controls impossible. To overcome these problems, the Gal4 system has been modified to permit both spatial and temporal control of transgene expression by the use of fusions proteins containing the Gal4 DNA binding domain and steroid receptor ligand binding domains (Han et al., 2000; Osterwalder et al., 2001; Roman et al., 2001). We have made a new Drosophila vector system that is based on the Gene-Switch fusion protein (Wang et al., 1994). These vectors are designed to facilitate the rapid cloning of promoters and enhancers in front of the Gene-Switch coding region. We have used these vectors to generate lines in which Gene-Switch expression is controlled by the Glass Multiple Repeat enhancer (GMR) from the ninaE regulatory region (Ellis et al., 1993). These P{GMRGS} lines provide inducible, eye-delimited expression of UAS transgenes. The Gene-Switch fusion protein is composed of the Sacchromyces cerivisiae Gal4 DNA binding domain, the human progesterone receptor ligand binding domain, and the human p65 transcriptional activation domain (Burcin et al., 1998; Wang et al., 1994). Antiprogestins, such as RU486, will bind to the progesterone receptor ligand binding domain and activate the Gene-Switch molecule. Once activated, Gene-Switch will promote transcription from the UAS Gal4-regulated promoters. The Gene-Switch protein, when expressed in Drosophila, has almost undetectable activity in the absence of RU486; however, feeding RU486 to Gene-Switch expressing flies leads to a dramatic induction of transcription from Gal4-regulated promoters (Osterwalder et al., 2001; Roman et al., 2001). Gene-Switch is responsive to RU486 in a dose-dependent manner and feeding flies 500 M RU486 for 1 h appears to saturate the response (Osterwalder et al., 2001; Roman et al., 2001). GeneSwitch-dependent activation can be detected in as little as 3 h and reaches maximum levels at approximately 24 h after the initial feeding. Thus, the timing and level of transcription of a UAS transgene can be manipulated through the controlled feeding of RU486. We have previously used enhancer detector constructs to achieve spatially restricted expression of the Gene-Switch fusion protein in a number of tissue and cell types (Roman et al., 2001). Additionally, both the Myosin Heavy Chain and elav promoters have been fused to the Gene-Switch coding sequences to provide, respectively, muscle and nervous system-specific expression in Drosophila (Osterwalder et al., 2001). Nevertheless, there remain many interesting and important cells and tissues for which we do not yet have Gene-Switch expressing lines. Further defined promoters and enhancers should be an important source for these desirable lines. Therefore, we have made a series of new P-elements that can be used to identify new transcriptional regulatory sequences through their ability to drive GeneSwitch expression. The new Gene-Switch P-elements allow for the rapid and efficient cloning of enhancers and promoters in front of the Gene-Switch coding sequences. A common problem with cloning into P-element vectors is the lack of unique restriction sites. Additionally, the need to clone many different DNA fragments into several different vectors can take a significant amount of time and effort in both the planning and cloning. The new vectors are an adaptation of the Univector plasmid-fusion system, which utilizes the cre-loxP site-specific recombination system of phage P1 (Liu et al., 1998). The Univector P-element system is composed of a donor plasmid and an acceptor P-element (Fig. 1). After the enhancer or promoter sequence is cloned into the multiple cloning sequence of the donor plasmid, cre-mediated in vitro recombination will fuse the two plasmids such that the