Improved Transformation with Capped Transposase mRNA in Pest Insects.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in mosquitoes, as models for hard-to-transform insects, showed that suppling transposase as mRNA increased the average transformation efficiency in from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in with mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of transposase delivered as a plasmid did not improve the transformation efficiency in or the agricultural pest . We believe that the use of mRNA has strong potential for enhancing transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.