piggyBac-mediated Germline Transformation of the Malaria Mosquito Anopheles stephensi Using the Red Fluorescent Protein dsRED as a Selectable Marker

Abstract

It is estimated that every year malaria infects ϳ300 million people and accounts for the death of 2 million individuals. The Plasmodium parasites that cause malaria in humans are transmitted exclusively by mosquito species belonging to the Anopheles genus. The recent development of a gene transfer technology for Anopheles stephensi mosquitoes, using the Minos transposable element marked with the enhanced green fluorescent protein EGFP (Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C., Kafatos, F. C., and Crisanti, A. (2000) Nature 405, 959 -962), provides now a powerful tool to investigate the role of mosquito molecules involved in the interaction with the malaria parasite. Such technology, when further developed with additional markers and transposable elements, will be invaluable for analyzing the biology of the vector and for developing malaria-resistant mosquitoes to be used as a tool to control malaria transmission in the field. We report here the germline transformation of A. stephensi mosquitoes using a piggyBac-based transposon to drive integration of the gene encoding for the red fluorescent protein dsRED. A. stephensi embryos were injected with transformation vector pPBRED containing the dsRED marker cloned within the arms of piggyBac. Microscopic analysis of G 1 larvae revealed the presence of seven fluorescent phenotypes whose different molecular origins were confirmed by Southern blotting analysis. Sequencing of the insertion sites in two lines demonstrated that integrations had occurred at TTAA nucleotides in accordance with piggyBac-mediated transpositions.The recent development of an efficient gene transfer technology for Anopheles stephensi mosquitoes, achieved by using a Minos-based transposon (4) loaded with the EGFP 1 selectable marker (1), has expanded the possibility of studying the genetics of human malaria vectors at the functional level. Although EGFP has proven to be an invaluable visible marker for identifying transformed individuals in different insect species (1, 5-7), the availability of only this selectable marker limits the range of applications of a gene transfer technology in malaria vectors. New molecular tools are now needed to exploit fully the potential of germline transformation for Anopheles mosquitoes, with the aim of unraveling the interactions between host molecules and Plasmodium parasites, as well as performing functional studies such as transposon tagging and enhancer trapping. Ultimately this technology could be used to develop transgenic mosquitoes with a nonpermissive phenotype for parasite development. These mosquitoes could be used in malaria control programs with the aim to replace the permissive wild type vectors.As shown in Drosophila melanogaster, the availability of various molecular and genetic tools to achieve germline transformation has contributed tremendously to our understanding of the fruit fly biology, leading to the identification of hundreds of genes involved in development, immunity, tissue modeling, and embryogenesis. The tran...

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“…The ingested blood meal (BM) is also visible. and 4% for the following combinations: A. stephensi with piggyBac (14), A. aegypti with Hermes (5, 15, 16), and A. aegypti with mariner (5,17).…”

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“…aegypti (Kokoza et al 2001), An. stephensi (Nolan et al 2002), An. albimanus (Perera et al 2002), An.…”

“…red fluorescent protein (dsRED) (Matz et al 1999) was also successfully employed in the transformation of An. stephensi (Nolan et al 2002).…”

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“…Because of its wide functionality in many insect species, its high transformation rates (up to 60% in Coleoptera, Berghammer et al 1999) and the perfect integration (Grossman et al 2000(Grossman et al , 2001Kokoza et al 2001;Nolan et al 2002) piggyBac seems to have advantages over the others cited above.…”