Developing biological systems can be approximately described as complex, three dimensional cellular assemblies that change dramatically across time as a consequence of cell proliferation, differentiation and movements. The presented project aims to overcome problems of limited resolution in both space and time of classical analysis by in situ hybridization on fixed tissue. The employment of the newly developed Single Plane Illumination Microscopy (SPIM) combined with new approaches for in vivo data acquisition and processing promise to yield high-resolution four-dimensional data of the complete Drosophila embryogenesis. We developed a toolkit for high-throughput gene engineering in flies, that provides means for creating faithful in vivo reporters of gene expression during Drosophila melanogaster development. The cornerstone of the toolkit is a fosmid genomic library enabling high-throughput recombineering and φC31 mediated site-specific transgenesis. The dominant, 3xP3-dsRed fly selectable marker on the fosmid backbone allows, in principle, transgenesis of the fosmid clones into any non-melanogaster species. In order to extend the capabilities of the gene engineering toolkit to include “evo-devo” studies, we generated genomic fosmid libraries for other sequenced Drosophilidae: D. virilis, D.simulans and D. pseudoobscura. The libraries for these species were constructed in the pFlyFos vector allowing for recombineering modification and φC31 transgenesis of non-melanogaster genomic loci into D. melanogaster. We have developed a PCR pooling strategy to identify clones for a specific gene from the libraries without extensive clone sequencing and mapping. The clones from these libraries will be primarily used for cross-species gene expression studies. As another application, transgenes originating from closely related species can be used to rescue D. melanogaster RNAi phenotypes and establish their specificity. Together with SPIM microscopy, the toolkit will allow to visualize gene expression patterns throughout Drosophila development.