Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects

Abstract

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac 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 Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes

“…aegypti line V19 contains one copy of the piggyBac construct pB_attp_3xP3-eGFP_attPrev. When homozygous, it shows strong eGFP expression in the eyes and in the ventral nerve cord, which has been published previously [89].…”

“…Preblastodermal embryos of the V19 eGFP-expressing transgenic line (pB_attp_3xP3-eGFP_attprev) of Ae. aegypti [89] were collected by allowing females homozygous for the single-copy transgene construct to oviposit for 30 min. Eggs were then rowed on a wet filter paper to ensure that all were in the same anterior-posterior orientation, transferred to double-sided sticky tape on a cover slide, and covered with halocarbon oil 27 (Sigma Aldrich/Merck KG, Darmstadt, Germany).…”

“…Then, the fold changes in all experimental replicates of one target gene or control treatment were averaged, and the standard deviation was calculated. Moreover, the transgene copy number of each larva was confirmed to be two by digital droplet PCR according to the protocol in [89]. The mosquito strains used for the following experiments were the Ae.…”