AUTHOR=Heryanto Christa , Mazo-Vargas Anyi , Martin Arnaud 

TITLE=Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

JOURNAL=Frontiers in Genome Editing

VOLUME=4

YEAR=2022

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2022.1074888

DOI=10.3389/fgeed.2022.1074888

ISSN=2673-3439

ABSTRACT=<p>While <italic>piggyBac</italic> transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized <italic>hyperactive piggyBac</italic> transposase (<italic>hyPBase</italic>) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth <italic>Plodia interpunctella</italic>. Co-injection of <italic>hyPBase</italic> mRNA with donor plasmids successfully integrated 1.5–4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the <italic>3xP3</italic> promoter. Somatic integration and expression of the transgene in the G<sub>0</sub> injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G<sub>0</sub> founders transmitted single-insertion copies of <italic>3xP3::EGFP</italic> and <italic>3xP3::EYFP</italic> and generated stable isogenic lines. Random in-crossing of a small cohort of G<sub>0</sub> founders expressing <italic>3xP3::DsRed</italic> yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how <italic>hyPBase</italic> can be used to generate stable transgenic resources in <italic>Plodia</italic> and other moths.</p>