Abstract
Transposon-mediated transgenesis has been widely used to study gene function in Lepidoptera, with piggyBac being the most commonly employed system. However, because the piggyBac transposase originates from a lepidopteran genome, it raises concerns about endogenous activation, remobilization, and silencing of transgenes, thus questioning its suitability as an optimal tool in Lepidoptera. As an alternative, we evaluated the dipteran-derived Minos transposase for stable germline transformation in the pantry moth, Plodia interpunctella. We injected syncytial embryos with transposase mRNA, along with donor plasmids encoding 3xP3::EGFP and 3xP3::mCherry markers of eye and glial tissues. Across multiple experiments, we found that G(0) injectees could transmit Minos transgenes through the germline even in the absence of visible marker expression in the soma, and that large mating pools of G(0) founders consistently produced transgenic offspring at efficiencies exceeding 10%. Using these methods, we generated transgenic lines with a dual expression plasmid, using 3xP3::mCherry for driving red fluorescence in eyes and glial tissues, as well as the Fibroin-L promoter expressing the recently developed mBaoJin fluorescent protein in the silk glands. This demonstrated the feasibility of screening two pairs of promoter activity in tissues of interest. Collectively, these results-along with previous findings in the silkworm Bombyx mori-demonstrate that Minos achieves robust germline integration of transgenes in Lepidoptera, offering a valuable pathway to the genetic modification of species where the remobilization or suppression of piggyBac elements might be rampant.