Site-specific integration and expression of a Plasmodium falciparum resistance transgene in Anopheles stephensi

We used the phi C31 site-specific integration system to produce transgenic Anopheles stephensi lines that express two effector molecules designed to target the human malaria parasite Plasmodium falciparum. These effector molecules are composed of an antimicrobial peptide, An. gambiae Cecropin A, joined to a single-chain antibody (scFv) derived from a P. falciparum-specific monoclonal antibody. The M4B7 immunotoxin contains an scFv designed to recognize Pfs25, a surface protein expressed by ookinetes, while the M2A10 immunotoxin contains an scFv designed to recognize circumsporozoite protein, a protein expressed on the surface of sporozoites. Previously characterized Anopheles cis-acting DNA regulatory elements were included in the transgene to coordinate immunotoxin production with parasite development. While the An. gambiae carboxypeptidase gene regulatory elements stimulate M4B7 expression in females within the first 12 hours post blood meal (hPBM), the An. stephensi vitellogenin gene regulatory elements direct expression of M2A10 in females ~12-24 hPBM. Through Southern blot, fluorescent hybridization in situ, RT-PCR, and western blot analyses, we confirmed transgene integration and expression. Having produced four transgenic lines that each contain a single copy of the M4B7/M2A10 transgene integrated into a different genomic location, we were able to observe the affect of flanking genomic DNA upon expression of these two immunotoxin genes.