Long-range targeted manipulation of the Drosophila genome by site-specific integration and recombinational resolution

Abstract

Significant advances in genomics underscore the importance of targeted mutagenesis for gene function analysis. Here we have developed a scheme for long-range targeted manipulation of genes in the Drosophila genome. Utilizing an attP attachment site for the phiC31 integrase previously targeted to the nbs gene, we integrated an 80-kb genomic fragment at its endogenous locus to generate a tandem duplication of the region. We achieved reduction to a single copy by inducing recombination via a site-specific DNA break. We report that, despite the large size of the DNA fragment, both plasmid integration and duplication reduction can be accomplished efficiently. Importantly, the integrating genomic fragment can serve as a venue for introducing targeted modifications to the entire region. We successfully introduced a new attachment site 70 kb from the existing attP using this two-step scheme, making a new region susceptible to targeted mutagenesis. By experimenting with different placements of the future DNA break site in the integrating vector, we established a vector configuration that facilitates the recovery of desired modifications. We also show that reduction events can occur efficiently through unequal meiotic crossing over between the large duplications. Based on our results, we suggest that a collection of 1200 lines with attachment sites inserted every 140 kb throughout the genome would render all Drosophila genes amenable to targeted mutagenesis. Excitingly, all of the components involved are likely functional in other eukaryotes, making our scheme for long-range targeted manipulation readily applicable to other systems.

Figure 1

Generating an 80-kb duplication by site-specific integration. (A) Integration schematic. At the top is the pWalkman vector built on the pP[acman] backbone, which contains P-element ends (solid arrowheads), a white maker (open oval), FRT sites (half-arrows), and an I-SceI cut site (IS1 in construct pWalkman{nbs-Ilp} IS1 and IS2 in pWalkman{nbs-Ilp} IS2). Below the pWalkman vector is the 82-kb nbs-Ilp insert, which contains two attB sites (solid arrows), one at each locus, separated by 68 kb. Below the 82-kb insert is the chromosomal region with an attP (shaded arrow) targeted to nbs. Note that the chromosomal region of Ilp does not contain any att site (wt). The positions for two sets of PCR primers (solid and shaded half arrows) used in B are indicated. A phiC31-mediated recombination (“X”) between attP and attB at nbs gives rise to the duplication depicted at the bottom. Only the integration of pWalkman{nbs-Ilp}IS1 is shown. The FRTs are not shown but are in close proximity to the P-element ends. (B) Representative results for diagnostic PCR tests. Marker size in kilobases is indicated to the left of the gel images. (Top) PCR analyses with nbs primers. The PCR templates are the following: lane 1, a wild-type line without any attachment site; lane 2, a duplication line from previous plasmid integration at attP@nbs; lane 3, plasmid DNA of pWalkman(nbs-Ilp)IS1; lane 4, the attP@nbs starting line; and lane 5, an integration line with the 80-kb duplication. (Bottom) Results of PCR analyses with Ilp primers. Lane “m” contains the marker. The PCR templates are the following: lane 6, a wild-type line, and lane 7, an integration line from this study.

Figure 2

I-SceI-induced reduction. (A) Reduction schematic. At the top is the 80-kb duplication resulting from integration of pWalkman{nbs-Ilp}IS1. The I-SceI endonuclease creates a DSB at IS1. The two nbs-Ilp regions align with three possible positions of recombination (“X”). The repair of the DSB gives rise to four types of events diagrammed below, with the first three being the product of recombination between the duplicated copies. The numbers in circles for the potential recombination sites correspond to the resulting types of reduction products. The positions of PCR primer sets are indicated (half arrows). In the NHEJ-type product, the white gene is disrupted (“broken” oval). The primer positions for “vector PCR” in B are indicated as half arrows. (B) Representative results for diagnostic PCR tests. The PCR templates for the top panels are the following: wt for lanes 1 and 5; a duplicated line for lanes 2 and 6; a type 3 reduction for lanes 3 and 7; a type 1 reduction for lane 4; and a type 2 event for lane 8. The 0.7-kb marker is indicated between the gel pictures. The PCR templates for the bottom panel are a reduction line for lane 9 and different NHEJ-type lines for lanes 10–16. Lane “m” represents markers with size indicated to the left in kilobases.

Figure 3

Spontaneous products are crossovers between the two duplicated copies. The two parental copies of the nbs-Ilp region are shown on the top with the four pieces of sequence heterology labeled according to their approximate distance from the left end of the nbs-Ilp region. The physical distance between heterologous sites is shown in kilobases below the diagram for the parental copies. (Bottom) Another representation of the nbs-Ilp parental copies and their five crossover products with each heterology depicted as a solid circle. The three types of reduction product have been defined in Figure 2. The number to the right of each product represents the number of lines identified for each outcome.

Figure 4

The effects of different repair mechanisms on reduction outcomes. (A) SSA repair as the mechanism for reduction. (Top) A DSB has been induced between two direct repeats (block arrows) with each horizontal line representing a DNA strand and vertical lines representing base pairing. The two heterologies between the repeats are denoted by an “X” and by an asterisk. The heterology indicated by the asterisk is more likely to become single stranded and thus excluded from the final reduction product due to its being closer to the DSB. (B) The “one-ended invasion crossover” model as the mechanism for reduction. The 80-kb duplication is shown for both pWalkman{nbs-Ilp} constructs. In the top diagram, IS1 is cut and the right end of the DSB in the bottom copy invades the homologous sequences of the top copy, leading to loss of the attB heterology. In the bottom diagram, IS2 is cut and the left end of the DSB in the top copy invades homologous sequences of the bottom copy, leading to retention of the attB. Note that in either case the invaded copy can be located on the sister chromatid instead.