|Handler, Alfred - Al|
Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2009
Publication Date: 10/27/2009
Citation: Schetelig, M.F., Scolari, F., Handler, A.M., Kittelmann, S., Gasperi, G., Wimmer, E.A. 2009. Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata. Proceedings of the National Academy of Sciences. 106(43):18171-18176. Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs is a major goal of our laboratory at the USDA, ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL. Development of this methodology and strategies to effectively and safely utilize transgenic insects for biological control will depend upon new ways to move genes and prevent their further movement. This article describes the development of a new gene transfer system. Transgenic strains can be comprehensively analyzed and tested for host strain fitness and optimal target strains can then be used for efficient integrations. Integrations can then be subsequently stabilized. This vector targeting and stabilization system was tested in the Mediterranean fruit fly Ceratitis capitata, and its use can be extended to other insects. This should provide a significant improvement for the development and safety of transgenic insect strains intended for field release.
Technical Abstract: Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to make the insertion homozygous, and lack of fitness costs, it may be advantageous to use that location for further modification and improvement. Here we describe an efficient method for the modification of previously inserted transgenes by the use of the site-specific integration system from phage phiC31 in the tephritid pest species, Ceratitis capitata (Mediterranean fruit fly or medfly). First, attP landing sites within transposon-based vectors were integrated randomly within a genome, with transgenic strains molecularly characterized. Secondly, donor plasmids containing a attB site, with additional markers, and transposon ends were integrated into the attP site by phiC31 integrase-mediated recombination. Next, transposase-encoding ‘jumpstarter’ strains were created and mated to transgenic strains resulting in the post-integrational excision of transposon ends, which left stably integrated transgene insertions that could not be remobilized. This three-step integration and stabilization system will allow the combination of several transgene-encoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental risks.