Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure.These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species.The genetic manipulation of insect genomes may herald novel strategies for the control of insect-borne disease and could provide the means both to limit economic damage by crop pests and increase productivity in commercially important insects. Such manipulation is now considered routine in the fruit fly, Drosophila melanogaster and is based on the exploitation of transposable genetic elements such as P. Current attempts at the transformation of non-drosophilid insects have also focused on this approach but phylogenetic restriction in mobility of the P-element has necessitated a search for alternative functional transposons [1]. As a result, there are now four transposable elements, derived from different eukaryotic transposable