Recombinase-mediated-cassette-exchange (RMCE) as a functional tool to study diapause in Aedes albopictus

Diapause is an anticipatory form of dormancy that is crucial to the overwinter survival of a wide range of insects. Transcriptomic techniques have revealed thousands of differentially expressed genes that are candidates for diapause regulation. However, functional validation with respect to diapause is an ongoing challenge because many candidate genes regulate processes throughout development. Therefore, manipulation of gene expression in a tissue and development-specific manner is critical to determining gene function in the context of diapause. Recombinase-mediated-cassette-exchange (RMCE) permits such precise manipulation by integrating transgene constructs at defined genomic regions (“landing-sites”). Since the transgene can be driven by a tissue or development-specific promoter, the RMCE system allows conditional regulation of gene expression.
We are developing the RMCE system to study diapause in Aedes albopictus, a widespread and invasive vector mosquito. We have generated multiple transgenic “landing-site” lines containing lox-recombination sequences. In a subsequent transformation, we targeted the lox-sites with a transgene, demonstrating the feasibility of recombination. In future transformations we will integrate transgene constructs that drive expression of hairpin RNAi sequences using a conditional promoter.
Previous work to understand the physiological and transcriptional basis of diapause suggest a role for maternal provisioning of lipids. Based on these studies, we hypothesize that fatty acid synthase (fas), a key enzyme during lipid synthesis, is part of the transgenerational signaling process to regulate diapause. To test the function of fas in a diapause-specific manner, we will conditionally manipulate fas using the RMCE system.