Site-specific transformation of Drosophila via phiC31 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 C31 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, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.

Figure 1.

Site-specific integration via φC31-mediated RMCE. (A) The target mini-white cassette is flanked by inverted attP sites within a genomic P element, and the donor yellow cassette is flanked by inverted attB sites on a plasmid. Thick solid line, transcription unit; shaded rectangles, P-element ends (not to scale); open triangles, att sites (not to scale). In this study, we use a 285-bp fragment from Streptomyces lividans for attB and a 221-bp fragment of φC31 for attP (Groth et al. 2000). Crossovers at both ends of the two aligned cassettes result in an exchange of mini-white for yellow. Alternatively, a single crossover at one end of the aligned cassettes creates an intermediate that integrates the entire donor plasmid and thus carries both yellow and mini-white. The single crossover can also occur at the other attP/attB pair, resulting in a related structure in which mini-white is to the left of yellow (not shown). Either intermediate may then resolve into an RMCE event by a subsequent crossover between the remaining attP/attB pair. (B and E) Representative flies from parental lines 42A and 52D carrying the target mini-white cassette. (C and F) Flies resulting from a single crossover (intermediate event). The pigmentation of the wings, abdominal stripes, and bristles of these flies is darker compared with that of the parental lines (C vs. B and F vs. E). In addition, note the increased eye pigmentation relative to that of the parental lines. (D and G) Flies representing RMCE events.

Figure 2.

Molecular analysis of RMCE candidates. (A) The structure of a yellow transgene resulting from an RMCE event. Each primer pair (arrows) used for PCR analyses included a primer that was unique to one end of the P element and a second primer in attR. Symbols are as defined for Figure 1. (B) Representative 5′ and 3′ PCR products from independent lines derived by RMCE from parental lines 25C and 42A. The expected 372-bp and 668-bp products were observed for all 43 lines tested. (C) Recombination between the attP (red sequence) and attB (blue sequence) sites occurs at a shared TTG to produce an attR (shown) and attL (not shown) site. Only sequences immediately flanking the core TTG are shown. PCR products, covering both recombination sites and representing 10 RMCE-derived lines from the 25C and 42A parental lines, were sequenced and found to contain the expected crossover.

Figure 3.

Southern analysis of RMCE-derived insertions of the yellow gene. (A) The two possible orientations of yellow following RMCE-mediated insertion into the genome. Restriction sites used for two separate genomic DNA digests and the expected fragments resulting from these digests are shown (B, BstXI; N, NdeI). * indicates the region (bar on top) homologous to the probe; all other symbols are as defined for Figure 1. (B) Southern analyses of five independent RMCE-derived lines from the 25C parental line. The 8.4- and 1.5-kbp bands from the BstXI digestion are weakly labeled by the probe. Both orientations of yellow were detected.

Figure 4.

Southern analysis of RMCE-derived insertions of GFP. (A) The two possible orientations of GFP following RMCE-mediated insertion into the genome. Restriction sites used for two separate genomic DNA digests and the expected fragments resulting from these digests are shown (S, SacI; C, ClaI). * indicates the region (bar on top) homologous to the probe; all other symbols are as defined for Figure 1. (B) Southern analyses of six unique RMCE-derived lines from the parental 42A target line. The 0.9- and 3.5-kbp bands resulting from the SacI digest are only weakly labeled by the probe. Both orientations of GFP were detected.