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Stable transformation and cloning mediated by piggyBac vector and RNA interference knockdown of Drosophila ovarian cell line

  • Hiroshi Uetake
  • Kenji Oka
  • Yuzo Niki
Report

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.

Keywords

Transfection piggyBac vector RNAi egfp OSS 

Notes

Acknowledgments

We are grateful to Dr. T. Tamura, Dr. M. Hatakeyama, Dr. H. Sano, and Dr. M. Hashitani for providing reagents and to H. Watanabe for his critical reading of this manuscript. This work was partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas, “Regulatory Mechanism of Gamete Stem Cells.”

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Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

  1. 1.Department of Sciences, Faculty of ScienceIbaraki UniversityMitoJapan

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