A nuclear targeting approach enables efficient transgenesis in the milkweed bug Oncopeltus fasciatus

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Abstract
Insects show extreme diversity and have long intrigued biologists. Although gene editing and transgenesis should enable the use of almost any insect species for biological research, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus , we first generate a useful eye- and body-color mutant strain by CRISPR/Cas9-mediated genome editing. Using this strain, we develop an efficient piggyBac -mediated transgenesis system with a nuclear targeting approach. Incorporating 1× and 3× nuclear localization signals into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus . Taking advantage of the mutant strain and the efficient transgenesis system, we integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated integration and histone-labelled strains for live imaging. We further show that the Q system, a binary expression system, is functional in this species, paving the way for sophisticated genetic manipulations. Together, our results expand the genetic toolkit of O. fasciatus as a comparative model insect and provide a practical framework for developing efficient transgenesis in other non-traditional model organisms. Summary statement: We developed an efficient piggyBac transgenesis system in the milkweed bug with a nuclear targeting approach, expanding genetic tools for non-model insects.
Funding Information
  • Japan Society for the Promotion of Science

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