Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.
Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.
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DOI：
10.3390/ijms242015155
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发表时间：
2023-10-13
影响因子：
5.6
通讯作者：
Schetelig MF
中科院分区：
文献类型：
--
作者：
Häcker I;Rehling T;Schlosser H;Mayorga-Ch D;Heilig M;Yan Y;Armbruster PA;Schetelig MF
转基因昆虫的创制是昆虫遗传学和分子生物学的关键技术。在昆虫转基因中广泛使用的工具是piggyBac转座酶，导致基本上随机的基因组整合。相反，位点特异性重组酶允许转基因构建体靶向整合到特定基因组靶位点中。然而，由于转基因效率低，这两种策略往往面临限制。我们的目的是通过利用加帽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。作为难以转化的昆虫的模型，系统地比较了伊蚊中的转基因效率，表明将piggyBac转座酶作为mRNA供应使埃及伊蚊中的平均转化效率从质粒来源的不到5%增加到mRNA的约50%。在Ae中观察到类似的高活性。白纹伊蚊pBac mRNA的表达。在重组实验中没有观察到质粒和mRNA之间的效率差异。此外，作为质粒递送的piggyBac转座酶的高活性版本没有提高Ae中的转化效率。埃及伊蚊或农业害虫铃木果蝇。我们相信，mRNA的使用具有很强的潜力，提高piggyBac转化效率在其他蚊子和重要的农业害虫，如实蝇。
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.
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影响因子：
3.8
作者：
Eckermann, Kolja N.;Ahmed, Hassan M. M.;Wimmer, Ernst A.
通讯作者：
Wimmer, Ernst A.
影响因子：
1.9
作者：
Allen, ML;Handler, AM;Skoda, SR
通讯作者：
Skoda, SR
影响因子：
2.4
作者：
Horn, C;Wimmer, EA
通讯作者：
Wimmer, EA
DOI：
10.1073/pnas.1110628108
发表时间：
2011-09-27
影响因子：
11.1
作者：
O'Brochta, David A.;Alford, Robert T.;Harrell, Robert A., II
通讯作者：
Harrell, Robert A., II
影响因子：
3.5
作者：
Messina, Giovanni;Celauro, Emanuele;Marsano, Rene Massimiliano;Prozzillo, Yuri;Dimitri, Patrizio
通讯作者：
Dimitri, Patrizio