Generation of transgenic anopheline mosquitoes refractory to malaria parasite by using gene manipulation

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In collaboration with Prof.Crisanti, the head investigator cloned the promoter region of the Anopheles gambiae carboxypeptidase (AgCP) gene. Two plasmid constructs (cytoplasmic and secreted forms) encoding the 13.1 single-chain antibody linked to Shiva (13.1 scFv-Shiva) under the control of the AgCP promoter were made. The plasmid DNA harboring the cytoplasmic gene was injected into Anopheles stephensi embryos. Using fluorescent positive selection, 5 lines of transgenic mosquitoes were obtained. The 13.1 scFv-Shiva gene was abundantly expressed in the guts of transgenic mosquitoes. Expression of the 13.1 scFv-Shiva was gut-specific and reached peak levels at about 3 h post-blood ingestion. The AgCP promoter can be used to drive the expression of genes that hinder parasite development in the mosquito gut. To measure the consequences of the 13.1 scFv-Shiva transgene expression on the parasite development, we fed control and transgenic mosquitoes on the same infected mouse and measured the numbers of oocysts formed Oocyst formation in transgenic mosquitoes was indistinguishable from that in wild type mosquitoes. Another line expressing the secreted form of 13.1 scFv-Shiva is in progress of examining.Prof.Crisanti's group showed that bee venom phospholipsase inhibits malaria parasites development in transgenic mosquitoes (JBC 2002). They also showed that genetic modification of mosquitoes offers exciting possibility for controlling malaria, but success will depend on how transmission affects the fitness of modified insects (Science 2003).Prof.Sinden's group joined the projects on genomic sequence of rodent malaria parasites. The sequence information provides insight into Plasmodium biology and disease (Nature 2002).Thus, we have established technology and facility for generating transgenic mosquitoes. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in malaria transmission.