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Updated: May 27, 2026
Embryo Microinjection for Transgenesis in Drosophila
Published on: June 7, 2024
Stable transformation and cloning mediated by piggyBac vector and RNA interference knockdown of Drosophila ovarian
Hiroshi Uetake1, Kenji Oka, Yuzo Niki
1Department of Sciences, Faculty of Science, Ibaraki University, Mito, 310-8512, Japan.
Abstract:
An in vitro study is a powerful method for elucidating gene functions in cellular and developmental events. However, until date, no reliable in vitro transformation, cloning, or knockdown system has been reported for Drosophila cells, with the exception of S2 and Kc cells. In this study, we demonstrated that the piggyBac vector stably integrates donor DNA into ovarian somatic sheets derived from follicle stem cells. The transformed ovarian somatic sheet cells were easily cloned with a new piggyBac selection vector carrying enhanced green fluorescent protein and dihydrofolate reductase genes, egfp, and dhfr, respectively, in culture media containing methotrexate, an inhibitor of DNA synthesis. Donor egfp continued to be expressed at a high level in long-term culture. Furthermore, the translation of donor egfp was inhibited by treatment with double-stranded RNA derived from the target gene. The transfection and cloning methods mediated by the piggyBac vector would thus be useful for future analyses of gene functions in OSS cells and possibly be applicable to other Drosophila cell lines.
Insights
Researchers developed a new method for studying gene function in Drosophila ovarian somatic sheet cells. This piggyBac vector system enables stable DNA integration, cloning, and gene knockdown for in vitro research.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- In vitro studies are crucial for understanding gene function in cellular and developmental processes.
- Limited in vitro transformation, cloning, and knockdown systems exist for Drosophila cells, hindering research.
Purpose of the Study:
- To establish a reliable in vitro system for gene function analysis in Drosophila ovarian somatic sheet (OSS) cells.
- To develop methods for stable DNA integration, cloning, and gene knockdown in OSS cells.
Main Methods:
- Utilized the piggyBac vector for stable donor DNA integration into OSS cells derived from follicle stem cells.
- Employed a new piggyBac selection vector with enhanced green fluorescent protein (egfp) and dihydrofolate reductase (dhfr) genes for cloning.
- Used methotrexate in culture media for selection and double-stranded RNA for gene knockdown.
Main Results:
- Achieved stable integration and high-level expression of donor egfp in long-term cultured OSS cells.
- Successfully cloned transformed OSS cells using the egfp and dhfr selection markers.
- Demonstrated inhibition of egfp translation via double-stranded RNA, indicating successful gene knockdown.
Conclusions:
- The developed piggyBac vector-mediated transfection and cloning system is effective for OSS cells.
- This system provides a valuable tool for future gene function analyses in Drosophila OSS cells.
- The methodology may be applicable to other Drosophila cell lines, expanding research capabilities.
