An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

Abstract

Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain-and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit.etermining the function of most genes is a long-term goal in the postgenomic era. This enterprise was initiated many decades ago, much before DNA sequencing, with the numerous forward genetic screens that have been carried out in Drosophila (1) and in other model organisms (2). Such screens have attained an exquisite degree of sophistication, allowing very specific biological functions to be probed. However, forward genetic screens are unlikely to uncover the function of all genes because their activity could be masked by redundancy. Moreover, the function of many genes might be overlooked if they serve a subtle function not needed for viability but essential for fitness in the wild. This is likely to be the case for many brain functions. Homologous recombination technology has the potential to knock out every gene, although this technology is still very laborious (3). Transgenic RNAi is another reverse genetic approach that has a place in the postgenomic era (4), but it is limited by the fact that it usually causes incomplete knock down and that it is still relatively laborious because it requires the construction and validation of individual transgenic strains. As a complement to the loss-of-function assays, misexpression screens based on the GAL4 system (5) have also been very successful in uncovering the activity of many genes in specific tissues (6). Ideally, however, gain-of-function analysis should always be complemented by the loss-of-function phenotype.The pattern of expression can be an alternative starting point for a genetic screen. For example, our work on embryonic boundaries in Drosophila suggests that segmentally expressed genes involved in segmental groove formation remain to be discovered (7). Presumably, these genes have not been identified in the past because of redundancy. A screen based on expression patterns could identify these genes as long as subse...

Neuronal fiber tracts connecting the brain and ventral nerve cord of the early Drosophila larva

“…Ascending axons were visualized with the help of the Gal4 driver line Nc1-Gal4, which came out of a screen for larval brain specific Gal4 driver lines (Larsen et al, 2006), and which is almost exclusively expressed in neurons of the ventral nerve cord. Based on number and density of labeled neurons, it appears as if most, if not all neurons of the cord express the driver line.…”

“…Egg collections were done on yeasted apple juice agar plates (Ashburner, 1989). The following fly lines were used: 3741-Gal4 (Bloomington Stock Center); So-Gal4 (Chang et al, 2003); MzVum-Gal4; UAS-CD8-GFP (Landgraf et al 2003); ChAT-Gal4+UAS-GFP (Salvaterra and Kitamoto, 2001); Nc1-Gal4 (Larsen et al, 2006). …”

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

“…3A). Recombination between these mutant FRT sites is reported to be incompatible (Larsen et al, 2006), which is essential for preventing the excision of the intervening sequence in presence of a FLP recombinase (FLP).…”

Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages

“…On the other hand, gene trap introduces random integration of a fluorescence reporter into the genome and the transgenic embryos suitable for live imaging can be selected by microscopic screenings. Gene-trap screenings have been carried out in various model organisms [21][22][23] and have generated valuable resources for live imaging embryonic development. However, mouse gene traps have thus far been primarily designed for genetic loss-of-function studies and expression analysis in fixed (e.g., by LacZ reporter), but not in live, tissues [24,25].…”