Captured segment exchange: a strategy for custom engineering large genomic regions in Drosophila melanogaster

Abstract

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be "captured" as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.

Figure 1

Conceptual view of captured segment exchange. (A) Existing technologies that form the basis of captured segment exchange. (Left) In RMCE, a marker flanked by SSR recognition sequences (RS) in the genome can be exchanged for a gene of interest (GOI) that is flanked by compatible RS on an injected plasmid. (Right) Crossovers between FRTs at nearby positions on homologous chromosomes can create a deletion of the intervening sequence on a resulting recombinant chromosome. (B) Captured segment exchange combines principles of the two technologies in A. RS at nearby positions on homologous chromosomes can effectively capture the intervening sequence such that a corresponding sequence flanked by compatible RS on an injected plasmid can replace the captured segment on a recombinant chromosome. Hatched region indicates the engineered sequence. Diagrams are not to scale.

Figure 2

Two-step dual RMCE strategy for captured segment exchange at the tsh locus. In step 1, a P[acman] clone carrying an engineered tsh sequence is integrated upstream of the endogenous tsh gene via phiC31-mediated transgenesis using the attP insertion PBac(y⁺-attP-3B)VK00003b (VK3b-attP). The mini-white carried by P[acman] and yellow carried by VK3b-attP serve as markers such that successful transformants are y⁺ w⁺ in an otherwise y⁻ w⁻ background. In step 2, the endogenous tsh locus is deleted from a recombinant chromosome resulting from FLP-mediated crossing over between FRTs on the two homologs, leaving only the engineered sequence; this event also deletes all mini-white and yellow markers from the recombinant chromosome, allowing candidates to be identified by a y⁻ w⁻ phenotype. Not shown is a reciprocal chromosome resulting from the FRT exchange that will carry a tandem duplication of the captured sequence along with all yellow and mini-white markers. Diagrams are not to scale.

Figure 3

Recombineering strategy to generate a donor BAC for captured segment exchange of tsh. (A) Two “homology arms” (LA and RA) are generated by PCR and subcloned into GMR-P[acman] to create GMR-P[acman]-tsh1. The forward primer for LA incorporates a 40-bp attB sequence at its 5′ end, whereas the reverse primer for RA incorporates a 35-bp FRT sequence at its 5′ end such that the final PCR product is flanked by attP and FRT sites. Not shown is an additional 20 bp at the 5′ end of the forward RA primer that is complementary to the reverse LA primer, which was used to splice by overlap extension the two PCR fragments together prior to subcloning (Horton et al. 1990), and a recognition site for BamHI, which was used to linearize the plasmid for gap repair. (B) GMR-P[acman]-tsh1 is linearized between the homology arms and transformed into bacteria that carry a tsh genomic clone (BAC) and Red recombination functions. The homology arms direct repair of the linearized plasmid from the BAC, creating GMR-P[acman]-tsh50, which carries a 50-kb sequence corresponding to the captured segment flanked by attP and FRT sites. Diagrams are not to scale.

Figure 4

Cross scheme for FLP-mediated deletion of the endogenous tsh locus. Virgin females carrying a heat-shock-inducible FLP gene (70FLP) and f06252-FRT are crossed to males in which GMR-P[acman]-tsh50 was integrated into VK3b-attP (VK3b-tsh50). The resulting larvae are heat-shocked to activate FLP expression, and male progeny are mated individually to virgin females carrying a second chromosome balancer (CyO) and arbitrary dominant markers (Sp Bl L). In the F₂, male progeny carrying CyO are scored for y and w phenotypes, with y⁻ w⁻ indicative of completed exchange. All crosses were carried out in a y⁻ w⁻ background.

Figure 5

Confirmation of tshNC1 deletion following captured segment exchange. (A) Schematic showing relative positions of tshNC1, tshNC2, and confirmation primers NC1check1for (F) and NC1check1rev (R1) ∼10 kb downstream of the tsh transcription unit. Captured segment exchange using GMR-P[acman]-tsh50ΔNC1 as a donor effectively results in deletion of tshNC1 from the chromosome, leaving the ∼1-kb bacterial galK gene in its place. Diagrams are not to scale. (B) Ethidium-stained gel showing PCR products from templates where tshNC1 is unaltered (+) or deleted (Δ) and replaced with galK using purified BAC DNA or Drosophila genomic DNA as templates. Candidate flies homozygous for the recombinant chromosome carrying the deletion (right-most lane) show the predicted PCR product. A similar strategy was used to confirm deletion of tshNC2 (Figure S2).

Figure 6

Captured segment exchange via single-step RMCE. (A) Schematic showing homologous chromosomes carrying attP64 (top) and attP52 (bottom), which are separated by ∼40 kb. The chromosome carrying attP52 is marked by mutations in ru to the left of the insert and in e to the right. Following injection of a plasmid carrying attB sites, captured segment exchange will result in a recombinant chromosome that is ru⁺, e⁻, lacks yellow markers, and carries unmarked plasmid sequence in place of the deleted captured segment (the faded line traces the regions of the parental chromosomes and the injected plasmid that are found in the final recombinant chromosome). (B) Cross scheme for accomplishing the exchange depicted in A (see text for details).