Gene trap technology in the Asian malaria mosquito, Anopheles stephensi Liston

The remobilization of genetic elements in insects allows for the development of forward-genetic tools aimed at identifying regions of interest within an insect’s genome. Forward genetic tools have been developed for the model insect Drosophila melanogaster Meigen, and have also been developed for insects of medical importance, including mosquitoes. Recently, the transposon piggyBac has been shown to remobilize within the genome of the Anopheles stephensi Liston and genetic tools to identify enhancer elements have been established. In our work, we expanded on the genetic tools in An. stephensi to include a gene-trapping technology. Two gene-trapping elements were developed in An. stephensi, which can be remobilized within the genome when crossed with a piggyBac transposase expressing line of An. stephensi. The first of the two elements contained a phase-zero splice acceptor, which when inserted into a like-phased protein-coding gene, resulted in a fusion protein containing the fluorescent marker. The second element contained the same phase zero splice acceptor followed by a viral internal ribosomal entry sequence, to allow for expression of the fluorescent protein regardless of the phase of the introns of the target gene. The detectable frequency of element remobilization ranged from 0.25 to 1%, and resulted in tissue-specific marker expression, including expression in tissues such as muscle, neural, fat body, imaginal discs, and salivary glands. The gene trap technology in An. stephensi represents an exciting new tool for the identification of tissue specific gene expression in mosquitoes, as well as the potential for the establishment of gene knock-out lines, and possible non-coding RNA detection. This technology could be applied in insects in which germ-line transformation is available.