Site-Specific Transformation of Drosophila via ϕC31 Integrase-Mediated Cassette Exchange

Abstract

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the fC31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, fC31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs. B IOLOGICAL research is greatly facilitated by our ability to manipulate DNA sequences in vivo. In the model organism Drosophila melanogaster, exogenous sequences are routinely incorporated into the genome using the P-element transposon system developed by Rubin and Spradling (Rubin and Spradling 1982;Spradling and Rubin 1982). The harnessing of this technology afforded a new ability to alter the genome, allowing the construction of transgenic animals by the relatively simple method of embryonic injection. Furthermore, P elements have been indispensable as a platform for developing new technologies for Drosophila research including the GAL4/UAS system (Brand and Perrimon 1993), targeted deletions (Ryder et al. 2004), and high-resolution mapping of mutations (Zhai et al. 2003).Recently, strategies have been developed to repeatedly incorporate transgenes into a single position in the genome. This provides a significant advantage over conventional P-element transformation, which occurs in an untargeted fashion and therefore subjects transgenes to varying position effects. In general these new strategies make use of site-specific recombinases, which catalyze crossovers between defined target sequences (Branda and Dymecki 2004). The most popular of these enzymes are FLP and Cre, which are widely used for many applications in Drosophila, including clonal analysis, targeted deletions, and tissue-specific excision (Golic and Lindquist 1989;Golic 1991; Hartl 1996, 2000;Heidmann and Lehner 2001;Ryder et al. 2004). A simple form of transgene targeting using these enzymes involves P elements carrying two transgenes, one flanked by loxP target sites for the Cre recombinase and the other flanked by FRT target sites for the FLP recombinase. Once such a P element integrates, subsequent treatment with Cre or FLP excises one or the other transgene, effectiv...

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

“…A further example for expandability of this system is the ability to make iterative in vivo assemblies (26), which will permit the analysis of large DNA units at a given locus. The elimination of vector sequences from integrating constructs, an advantage usually claimed by recombinasemediated cassette exchange (RMCE) approaches (8,9,27), can also be achieved with our system by Cre-mediated excision, leaving essentially only one loxP site and the transgene with its regulatory elements at the genomic locus (see Figs. 1c and 3c).…”

Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

“…The phiC31 integrase mediates recombination between the bacterial and phage attachment sites, attB and attP, and has been shown to efficiently integrate attB-containing plasmids into attP 'landing sites' that have been previously inserted in the genome 22 . To date, over 100 attP landing-site loci have been randomly integrated into the Drosophila genome [22][23][24][25] . Some of these landing sites have been modified to allow for recombinase-mediated cassette exchange 23 and the unambiguous detection of integrated transgenes 25 , whereas others have been shown to be amenable to the integration of large 100-kb DNA constructs 24 .…”

“…To date, over 100 attP landing-site loci have been randomly integrated into the Drosophila genome [22][23][24][25] . Some of these landing sites have been modified to allow for recombinase-mediated cassette exchange 23 and the unambiguous detection of integrated transgenes 25 , whereas others have been shown to be amenable to the integration of large 100-kb DNA constructs 24 . However, it remains unclear whether position effects at any of these attP sites permit optimal transgene expression.…”

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

“…We used the phiC31 landing pad system, where phiC31 integrase mediates recombination between a landing pad (inserted in the genome) containing phage attachment (attP) sites and a donor construct containing bacterial attachment (attB) sites (5). This system allows us to maintain a constant genomic context for multiple transgenic 1360 sequence constructs.…”