Site-specific genomic targeting inDrosophila

Abstract

Transcriptional regulation of transgenes depends upon genomic localization in higher eukaryotes. For the applied use of transgenic organisms as producers of pharmaceutically relevant proteins or as pest population control agents, a method to make transgene expression predictable is highly desirable. A targeting method that allows precise cassette replacement comprising solely genes of interest (without extraneous donor vector sequences) would be highly advantageous for insects and other multicellular organisms. In this report, we describe a method for transgene targeting to predefined chromosomal sites in Drosophila by using a transposon vector that, once integrated in the germ line, acts as an acceptor site for donor vectors. To make recombinational insertions irreversible, a FLP recombinase-mediated cassette exchange strategy was used, and to enhance donor-target pairing, a homing sequence from the linotte locus was used. Site-specific recombinants were screened by interconvertible eye fluorescence marker phenotypes yielding, on average, targeted insertions at a frequency of 23%. The cassette exchange system provides for repetitive integrations into the same locus, allowing comparative analysis of true transgenic alleles. Furthermore, this method was used to stabilize a targeted transgene by the postintegration excision of putatively mobile transposon sequences. The genomic targeting and stabilization strategy described for Drosophila should be applicable to other insects, specifically for the goals of optimizing heterologous protein expression and enhancing ecological safety of transgenic strains intended for release in biocontrol programs.