With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed ''genomic engineering'' in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase C31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knockout line is generated by deleting the target gene and replacing it with an integration site of C31. Second, DNA integration by C31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles.cell polarity ͉ ends-out targeting ͉ homologous recombination ͉ phiC31 integrase T he development of homologous recombination (HR)-based gene targeting was a major breakthrough in Drosophila genetics (1, 2). At present, in Drosophila as well as in mice, a HR-based approach is virtually the only way to make directed modifications of a target gene (3, 4). However, because the entire targeting process must be repeated for making each allele, the amount of time and labor may become impractical to make more than just a few targeted alleles. In addition, because of the requirement of HR, it can be very difficult to introduce appreciably complicated DNA sequence modifications by gene targeting. The current lack of adequate genetic tools for directed and efficient modifications of the genome presents a major hurdle in Drosophila genetics today. For example, many of the protein pathways that are highly conserved between Drosophila and vertebrates, such as the cell polarity pathway (5), appear to be exceedingly complex. Rigorous genetic dissections of such intricate protein networks can be highly challenging, because in most cases the functions of mutated or modified individual genes of such pathways can only be assayed by artificial over-expression methods, which often lack the requisite controllability and fidelity of gene expression. One ideal solution would be for each protein gene of interest to generate, at the gene's native genomic locus, a set of defined mutant alleles that are strategically designed to test hypotheses about the protein's in vivo functions and interactions. Furthermore, being able to generate any conceivable alleles of a target gene, such as functional fusion alleles of fluorescent proteins/purification tags or alleles with conditional activities, would also offer us unprecedented freedom and opportunities to explore unique experiments of imaging, proteomics, and disease models.To achieve the goal of such directed, efficient, and versatile modifications of the Drosophila genome, we have developed a...

Wings are essential for insect fitness. A number of proteins and enzymes have been identified to be involved in wing terminal differentiation, which is characterized by the formation of the wing cuticle. Here, we addressed the question whether chitinase 10 (Cht10) may play an important role in chitin organization in the wings of the fruit fly Drosophila melanogaster. Initially, we first found that Cht10 expression coincides with the expression of the chitin synthase coding gene kkv. This suggests that the respective proteins may cooperate during wing differentiation. In tissue-specific RNA interference experiments, we demonstrate that suppression of Cht10 causes an excess in chitin amounts in the wing cuticle. Chitin organization is severely disrupted in these wings. Based on these data, we hypothesize that Cht10 restricts chitin amounts produced by Kkv in order to ensure normal chitin organization and wing cuticle formation. In addition, we found by scanning electron microscopy that Cht10 suppression also affects the cuticle surface. In turn, cuticle inward permeability is enhanced in Cht10-less wings. Moreover, flies with reduced Cht10 function are unable to fly. In conclusion, Cht10 is essential for wing terminal differentiation and function.

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