Department of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, Germany.
Liebig Centre for Agroecology & Climate Impact Research, 35394 Giessen, Germany.
Int J Mol Sci. 2023 Oct 13;24(20):15155. doi: 10.3390/ijms242015155.
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.
生成转基因昆虫是昆虫遗传学和分子生物学的一项关键技术。转座酶是昆虫转基因技术中广泛使用的一种仪器，它导致基因组的整合基本上是随机的。相比之下，位点特异性重组酶允许将转基因构建体靶向整合到特定的基因组靶位点。然而，这两种策略由于转基因效率低，常常受到限制。我们的目标是通过利用 capped mRNA 作为转座酶或重组酶的来源，而不是辅助质粒，来提高转基因效率。我们系统地比较了作为难以转化昆虫模型的蚊子中的转基因效率，结果表明，以 mRNA 形式提供转座酶将平均转化效率从质粒来源的不到 5%提高到约 50%。在 中也观察到类似的高活性。在重组实验中，质粒和 mRNA 之间没有观察到效率差异。此外，作为质粒传递的超活性版本的 转座酶并没有提高 或农业害虫 的转化效率。我们相信，在其他蚊子和重要的农业害虫（如桔小实蝇）中，使用 mRNA 具有提高 转化效率的强大潜力。
