10] In Vivo analysis of lacZ fusion genes in transgenic Drosophila melanogaster

Abstract

Publisher Summary This chapter presents the in vivo analysis of lacZ fusion genes in transgenic Drosophila melanogaster . lacZ reporter genes have been used most successfully for unraveling the in vivo transcription mechanisms that control embryonic pattern formation. These studies combine genetic, biochemical, and transgenic experiments: By crossing a line containing a lacZ transgene into Drosophila mutants, regulatory genes involved in the activation and repression of the transgene can be identified. In many cases, these genes encode transcription factors, and DNA-binding studies are performed to determine whether they bind directly to sites within the enhancer. Individual binding sites are then subjected to in vitro mutagenesis, and lacZ reporter genes containing the mutant forms of the enhancer are tested in vivo . If an identified binding site is important for the function of the enhancer, this will be reflected in changes in its expression pattern. The analysis of lacZ transgenes requires an efficient method for introducing exogenous DNA into the genome. This was first accomplished by Rubin and Spradling, who took advantage of the transposition properties of Pelements, which are natural transposons found in some strains of Drosophila . Their work is the basis for the protocol for generating transgenic flies that is presented in the chapter.