Reconfiguring gene traps for new tasks using iTRAC

Abstract

We recently developed integrase-mediated trap conversion (iTRAC) as a means of exploiting gene traps to create new genetic tools, such as markers for imaging, drivers for gene expression and landing sites for gene and chromosome engineering. The principle of iTRAC is simple: primary gene traps are generated with transposon vectors carrying φC31 integrase docking sites, which are subsequently utilized to integrate different constructs into the selected trapped loci. Thus, iTRAC allows us to reconfigure selected traps for new purposes. Two features make iTRAC an attractive approach for Drosophila research. First, its versatility permits the exploitation of gene traps in an open-ended way, for applications that were not envisaged during the primary trapping screen. Second, iTRAC is readily transferable to new species and provides a means for developing complex genetic tools in drosophilids that lack the facility of Drosophila melanogaster genetics.

Figure 1

Transgenic approaches for generating markers, expression drivers, gene tags and mutants in Drosophila. Approaches are grouped according to two criteria: (A) whether they rely on random or targeted insertions and (B) whether they employ transgenes designed for a specific purpose, or ones that are efficiently reconfigured for multiple tasks. In the latter case, the ϕC31 integrase system allows transgene insertions to be exploited for new purposes in an open-ended way. iTRAC involves screening random insertions for gene traps and allows these to be adapted for diverse applications.

Figure 2

Schematic overview of iTRAC. Gene traps are generated with a Minos transposon vector (inverted repeats shown with white arrowheads) carrying a trapping cassette with a visible reporter gene. Subsequently, selected gene traps can be exploited to generate new genetic tools (different reporters, GAL4 drivers, knockdowns, gene fusions, etc.,) by integrating new effector constructs into the trapped locus. This is achieved using the ϕC31 integrase and cognate attP and attB sites present in the original trapping construct and in the effector constructs, respectively (black and gray arrows). Different configurations and possible uses of iTRA C are described in the text.