Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species

Area of Science:

• Molecular Biology
• Genetics
• Entomology

Background:

• Advanced genome editing tools are available, but DNA transposase improvement is crucial for transgenesis, especially in non-model organisms.
• Random insertional mutagenesis is essential for identifying stable transgene integration sites free from position effects.
• A hyperactive piggyBac transposase (hyPBase) was engineered from the widely used PBase, available in insect (ⁱhyPBase) and mammalian (^mhyPBase) codon-optimized versions.

Purpose of the Study:

• To evaluate the efficacy of hyPBase, irrespective of codon usage, in increasing germline transformation rates in insects.
• To assess the utility of hyPBase for generating transgenic strains in diverse insect orders, including agricultural pests and disease vectors.
• To investigate and clarify previously reported high sterility rates associated with hyPBase.

Main Methods:

• Utilized helper plasmids encoding hyPBase (both ⁱhyPBase and ^mhyPBase) in transgenesis experiments.
• Conducted experiments in the Mediterranean fruit fly (Ceratitis capitata), red flour beetle (Tribolium castaneum), and vinegar fly (Drosophila melanogaster).
• Compared transposition and germline transformation rates with the classical PBase.

Main Results:

• hyPBase significantly increased germline transformation rates by up to 15-fold compared to PBase across tested insect species.
• Successfully generated transgenic Tribolium castaneum strains with constructs affecting fitness and viability.
• Did not confirm previously reported high sterility rates associated with hyPBase (iPB7).

Conclusions:

• hyPBase is a highly effective genetic engineering tool for insect transgenesis, offering significantly improved germline transformation efficiency.
• The codon usage of hyPBase does not impede its effectiveness in insect transgenesis.
• hyPBase is recommended for generating transgenic strains in diverse insect orders, including those with limited genomic information.