Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

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. 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 a tephritid pest species, the Mediterranean fruit fly Ceratitis capitata. First, suitable transgenic strains with randomly integrated attP landing sites within transposon-based vectors were identified by molecular and functional characterization. Second, donor plasmids containing an attB site, with additional markers, and transposon ends were integrated into attP sites by phiC31 integrase-mediated recombination. Third, transposase-encoding 'jumpstarter' strains were created and mated to transgenic strains resulting in the postintegrational 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 concerns.