|Handler, Alfred - Al|
Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2005
Publication Date: 8/30/2005
Citation: Horn, C., Handler, A.M. 2005. Site-specific genomic targeting in Drosophila. Proceedings of the National Academy of Sciences. 102:12483-12488. Interpretive Summary: The creation of transgenic strains of ecomically important insects for the development of more effective biological control programs is major goal of our laboratory at CMAVE. Development of this methodology and strategies to effectively and safely utilize transgenic insects for biological control will depend upon new vector systems that can be targeted to specific genomic integration sites and then stabilized to minimize strain breakdown and ecological risks related to unintended movement of the transgenes to other organisms. This article describes the delelopment of a new gene transfer vector system that allows the creation of target site strain using piggyBac vectors that can be comprehensively analyzed and tested for host strain fitness and genomic effects on gene expression from the target site. Optimal target strains can then be used for efficient and repetitive integratibns by FRT recombinase-mediated cassette exchange. FRT integrations that carry a duplicated piggyBac terminus can be subsequently stabilized by terminus deletion. This vector targeting and stabilization system was tested in Drosophila, but its use can be extended to any insect subject to transposon and FRT-mediated genomic integration, and should provide a signifipant improvement for the development and safety of transgenic insect strains intended for field release.
Technical Abstract: Transcriptional regulation of transgenes is dependent 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. Currently, gene targeting mediated by site-specific recombinases is not available for many multicellular organisms, including insects. In this report we describe a novel method for transgene targeting to pre-defined chromosomal sites in Drosophila using a transposon vector that once integrated in the germline acts as acceptor site for donor vectors. To make recombinational insertions irreversible, a FLP recombinase mediated cassette exchange (RMCE) strategy was utilized and to enhance donor-target pairing a homing sequence from the linotte locus was employed. Site-specific recombinants were screened by interconvertible eye fluorescence marker phenotypes yielding, on average, targeted insertions at a frequency of 23%, verified by insertion site sequencing and Southern hybridization. Furthermore, this method was used to stabilize a targeted transgene by post-recombinational deletion 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.