A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting

We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site.We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.Recent progress in the development of transposon-mediated germline transformation in non-drosophilid insects has been dramatic. There are now four transposable element systems (Mos1-mariner, Hermes, Minos and piggyBac) that have been successfully deployed across a range of dipteran, lepidopteran and coleopteran insects [1]. This progress has served to focus attention onto potential applications of the technology and, as the number of transgenic strains continues to increase, consideration is being given to inherent problems of the approach and to potential alternatives. All transposon-based approaches to transformation are constrained by the quasi-random nature of the integration sites, which can give rise to insertional inactivation of essential genes and to position effects on transgene expression [2]. As an alternative approach, we have been investigating the potential for gene targeting in insect genomes thr