Abstract
Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.
Highlights
Despite intense efforts, malaria is globally responsible for almost one million deaths per year [1], the majority of which are in Africa
Control measures that focus on the vector remain the most effective and deployment of transgenic mosquitoes that are refractory to malaria transmission is increasingly seen as having great potential [4]
We reported the first site-directed transformation of mosquitoes using the phiC31 system in the arboviral vector Aedes aegypti [26] and subsequently demonstrated its utility for expression of an antimalarial effector transgene in An. gambiae [21]
Summary
Malaria is globally responsible for almost one million deaths per year [1], the majority of which are in Africa. Control measures that focus on the vector remain the most effective and deployment of transgenic mosquitoes that are refractory to malaria transmission is increasingly seen as having great potential [4]. This is true in regions such as subSaharan Africa where transmission rates are very high and existing interventions are not expected to be sufficiently effective [5]. The focus of this study is An. gambiae, the principal vector of malaria in endemic regions of Africa
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