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 . As a result, there are now four transposable elements, derived from different eukaryotic transposable element families, that have been successfully deployed across dipteran, lepidopteran and coleopteran insects. First, the Mosl element, derived from D. mauritiana and belonging to the Mariner family , which has been used to transform D. melanogaster, D. virilis, Aedes aegypti and Musca domestica. Secondly, the Hermes element, derived from the house fly M. domestica and a member of the hAT family , which has been used to transform D. melanogaster, Ae. aegypti, Anopheles gambiae cells , Tribolium castaneum, Stomoxys calcitrans, Ceratitis capitata and Culex quinquefasciatus [P. W. Atkinson, personal comm.]. Thirdly, the Minos element, derived from D. hydei and a member of the Tc1 family , which has been used to transform D. melanogaster, C. capitata and An. stephensi. Finally, the piggyBac element, derived from Trichoplusia ni and a member of the TTAA family [18, 19], which has been used to transform C. capitata, D. melanogaster, T. castaneum, Bombyx mori, Pectinophora gossypiella, Bactrocera dorsalis, Anastrepha suspensa, M. domestica, Lucilia cuprina [M. J. Scott, personal comm.], Ae. aegypti [M. J. Fraser, personal comm.], An. gambiae [M. Q. Benedict, personal. comm.], An. stephensi [M. Jacobs-Lorena, personal comm.] and An. albimanus [A. M. Handler, personal comm.].
Apart from this focus on transposable elements, other approaches to the transformation of non-drosophilid insects include the use of viral vectors. The Sindbis Alphavirus  is proving to be particularly effective at transducing genes into mosquito tissue  but does have certain limitations and has not been used successfully to generate transgenic insects. Similarly, pantropic retroviruses have been used to mediate stable gene transfer and gene expression in somatic cells from a variety of insect species [29–31] but have not proved effective at generating transgenic insects.
Despite these recent successes, both transposon and viral-mediated strategies are constrained to some extent by the quasi-random nature of the integration sites. This can give rise to insertional inactivation of essential genes and all transgenes introduced in this way can suffer dramatically from position effects on expression. For example, the transgene may not be expressed (or may be expressed sub-optimally) if integration occurs in a transcriptionally inactive region of the genome. As an alternative approach to insect transformation, we have been investigating the potential of gene targeting through homologous recombination. Such a mechanism would be independent of phylogenetic restrictions on transposon mobility and free of concerns over transgene instability mediated by non-specific transposases from endogenous mobile elements. Gene targeting would facilitate the precise introduction of transgenes into predetermined chromosomal sites of demonstrated transcriptional activity. It could also be used to 'knock out' both alleles of an endogenous gene in order to achieve specific phenotypic modifications. In addition, with appropriate construction of the gene-targeting vector, it would be possible to introduce specific mutations into a target gene and study the resulting phenotype  or revert mutant to wild-type alleles .
Gene targeting through homologous recombination has been exploited in yeasts , mammalian cells [35–38], protozoans [39–43], slime mould , plant cells , intact plants such as the moss, Physcomitrella patens and Arabidopsis, fungal pathogens [48, 49] and chicken cells . Although little information is available for insects, the investigation and optimisation of targeting strategies has been greatly facilitated by using cultured somatic cells as a model system. Through such approaches, the machinery of homologous recombination has been demonstrated in both mosquitoes  and Drosophila. More recently, gene targeting has been demonstrated in vivo in Drosophila through an elegant combination of transposon-mediated transformation and site-specific recombination. In these experiments, a construct carrying part of the target gene was integrated by means of a transposable element vector. Subsequently, a site-specific recombinase (FLP) and a site-specific endonuclease (I-SceI) were used to generate extrachromosomal DNA molecules with a double-strand break in the region of homology. Such molecules would be present in every nucleus, providing an efficient substrate for gene targeting .
The most significant progress in optimising experimental parameters for gene targeting has involved the use of mouse embryonic stem cells and these studies have revealed the importance of vector design . In particular, factors such as overall length of homology, isogenicity between donor and target sequences and vector topology may play an important role. Moreover, it has been found necessary to incorporate positive selectable markers to identify transformants as well as some mechanism for the enrichment of targeted integrations, which are likely to occur at a lower frequency than random (non-homologous) events. Such enrichment might include the use of negative selectable markers, such as the HSV-tk gene, which is cytotoxic in the presence of nucleoside analogues . Similarly, it may involve promoter-trap strategies where a positive selectable marker, such as a neomycin resistance gene, is only expressed from an endogenous promoter in the event of targeted integration .
As part of our attempts to define the potential of gene targeting in the mosquito, we describe here the successful targeting of a hygromycin resistance transgene, previously transformed into the Ae. aegypti Mos20 cell line and stably maintained as one or more multi-copy, extrachromosomal tandem arrays. The targeting replacement vector we employed carried a region of homology (the hygromycin resistance gene and SV40 terminator) disrupted by a promoterless neomycin resistance gene (neo). In this design, the neo gene serves as a promoter-trap for the enrichment of targeted integration since neomycin resistance can only be expressed from the promoter of the hygromycin resistance gene in the event of precise homologous recombination. Targeted integration events would therefore be detectable both by efficient expression of neomycin resistance and by inactivation (knockout) of hygromycin resistance.