Minos-mediated transgenesis in the pantry moth Plodia interpunctella

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 G₀ 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 G₀ 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.