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.
DROSOPHILA melanogaster is an excellent organism for the genetic analysis of cellular and developmental processes. For the past two decades, methods of gene targeting by homologous recombination have facilitated the engineering of crafted mutations (Gloor et al. 1991;Rong and Golic 2000;Rong et al. 2002;Gong and Golic 2003). The wealth of information provided by bioinformatic analyses has further aided geneticists in the design of mutations. Gene targeting in flies, however, is quite labor intensive, discouraging its use in systematic mutagenesis, which involves more than one targeted allele, although success stories have revealed the benefits of such approaches (Gronke et al. 2010;Spitzweck et al. 2010).Recently, several laboratories have demonstrated that, by combining homologous recombination with the efficient phiC31-mediated site-specific recombination method, the need for repetitive gene targeting of individual alleles to the endogenous locus can be eliminated (Gao et al. 2008;Huang et al. 2009;Weng et al. 2009;Iampietro et al. 2010). We developed the site-specific integrase-mediated repeated targeting (SIRT) method, in which an attP attachment site for phiC31 integrase is first targeted to the genome in the proximity of the locus of interest using gene targeting by homologous recombination. Further modifications are carried out by integration of a plasmid that carries the modif...