Improved <i>piggyBac</i> 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 <i>piggyBac</i> transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted i...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-10-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/20/15155 |
| _version_ | 1797573647104212992 |
|---|---|
| author | Irina Häcker Tanja Rehling Henrik Schlosser Daniela Mayorga-Ch Mara Heilig Ying Yan Peter A. Armbruster Marc F. Schetelig |
| author_facet | Irina Häcker Tanja Rehling Henrik Schlosser Daniela Mayorga-Ch Mara Heilig Ying Yan Peter A. Armbruster Marc F. Schetelig |
| author_sort | Irina Häcker |
| collection | DOAJ |
| description | Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the <i>piggyBac</i> 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 <i>Aedes</i> mosquitoes, as models for hard-to-transform insects, showed that suppling <i>piggyBac</i> transposase as mRNA increased the average transformation efficiency in <i>Aedes aegypti</i> from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in <i>Ae. albopictus</i> with <i>pBac</i> mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of <i>piggyBac</i> transposase delivered as a plasmid did not improve the transformation efficiency in <i>Ae. aegypti</i> or the agricultural pest <i>Drosophila suzukii</i>. We believe that the use of mRNA has strong potential for enhancing <i>piggyBac</i> transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids. |
| first_indexed | 2024-03-10T21:13:02Z |
| format | Article |
| id | doaj.art-b3a58a8cae2a4c6c965f70b102913528 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T21:13:02Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-b3a58a8cae2a4c6c965f70b1029135282023-11-19T16:42:25ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-10-0124201515510.3390/ijms242015155Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest InsectsIrina Häcker0Tanja Rehling1Henrik Schlosser2Daniela Mayorga-Ch3Mara Heilig4Ying Yan5Peter A. Armbruster6Marc F. Schetelig7Department of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyDepartment of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyDepartment of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyDepartment of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyDepartment of Biology, Georgetown University, 37th and O Streets NW, Washington, DC 20057-1229, USADepartment of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyDepartment of Biology, Georgetown University, 37th and O Streets NW, Washington, DC 20057-1229, USADepartment of Insect Biotechnology in Plant Protection, Justus Liebig University Giessen, Winchesterstr. 2, 35394 Giessen, GermanyCreating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the <i>piggyBac</i> 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 <i>Aedes</i> mosquitoes, as models for hard-to-transform insects, showed that suppling <i>piggyBac</i> transposase as mRNA increased the average transformation efficiency in <i>Aedes aegypti</i> from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in <i>Ae. albopictus</i> with <i>pBac</i> mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of <i>piggyBac</i> transposase delivered as a plasmid did not improve the transformation efficiency in <i>Ae. aegypti</i> or the agricultural pest <i>Drosophila suzukii</i>. We believe that the use of mRNA has strong potential for enhancing <i>piggyBac</i> transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.https://www.mdpi.com/1422-0067/24/20/15155insect transgenesis<i>Aedes</i><i>Drosophila suzukii</i>tephritidstransformation efficiencyrecombination efficiency |
| spellingShingle | Irina Häcker Tanja Rehling Henrik Schlosser Daniela Mayorga-Ch Mara Heilig Ying Yan Peter A. Armbruster Marc F. Schetelig Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects International Journal of Molecular Sciences insect transgenesis <i>Aedes</i> <i>Drosophila suzukii</i> tephritids transformation efficiency recombination efficiency |
| title | Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects |
| title_full | Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects |
| title_fullStr | Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects |
| title_full_unstemmed | Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects |
| title_short | Improved <i>piggyBac</i> Transformation with Capped Transposase mRNA in Pest Insects |
| title_sort | improved i piggybac i transformation with capped transposase mrna in pest insects |
| topic | insect transgenesis <i>Aedes</i> <i>Drosophila suzukii</i> tephritids transformation efficiency recombination efficiency |
| url | https://www.mdpi.com/1422-0067/24/20/15155 |
| work_keys_str_mv | AT irinahacker improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT tanjarehling improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT henrikschlosser improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT danielamayorgach improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT maraheilig improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT yingyan improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT peteraarmbruster improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects AT marcfschetelig improvedipiggybacitransformationwithcappedtransposasemrnainpestinsects |