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 transgeneencoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental concerns.insect pest management ͉ phiC31 integrase ͉ transgene stability

Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa

“…This is possible through phiC31 attP recombination sites placed within the driver and effector constructs that have been proven functional in fruit flies (19)(20)(21)(22) and mosquitoes (23,24). This technology could be used for stabilizing the transposon vector, by introducing a tandem duplication of one inverted terminal repeat sequence that allows post-integration deletion of the other terminus, thereby immobilizing remaining transgene sequences.…”

Not all GMOs are crop plants: non-plant GMO applications in agriculture

“…These vectors were stabilized by either mating to a jumpstarter strain in medfly, or injecting a transposase helper plasmid in the Anastrepha species. Stability in the presence of transposase was confirmed quantitatively in C. capitata by testing more than 70,000 progeny (Schetelig et al 2009). …”

“…Recently we tested a new Cre/lox RMCE system that showed function for the first time in a nondrosophilid species, A. suspensa, as well as in Drosophila (Schetelig and Handler 2013). The unidirectional phiC31 integrase recombination system (Groth et al 2004) has also been tested for site-specific attB/attP genomic targeting in C. capitata (Schetelig et al 2009), as well as in several mosquito species.…”

“…These vectors were first tested individually and then in combination in an integrated targeting/ stabilization system in Drosophila melanogaster Horn and Handler 2005). We have since modified these vectors for more efficient and flexible use in tephritid fruit fly species to test their function (Schetelig et al 2009), and have initiated the creation of a series of stabilized target-site strains that can be used to create transgenic strains for biological control and functional genomics analysis (Meza et al 2011). This new generation of transformation vectors is expected to increase the efficiency of transgenic strain development and strain effectiveness, while improving their ecological safety.…”

“…Modified stabilization vectors were subsequently created for tephritid species and tested in the medfly, Ceratitis capitata (Schetelig et al 2009), mexfly, Anastrepha ludens (Meza et al 2011), and caribfly, Anastreph suspensa (A. M. Handler, unpublished data). These vectors were stabilized by either mating to a jumpstarter strain in medfly, or injecting a transposase helper plasmid in the Anastrepha species.…”