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

  • Hiroshi Uetake
  • Kenji Oka
  • Yuzo NikiEmail author


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.


Transfection piggyBac vector RNAi egfp OSS 



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.”


  1. Bai J.; Binari R.; Ni J. Q.; Vijayakanthan M.; Li H. S.; Perrimon N. RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance. Development 135: 1439–1449; 2008.PubMedCrossRefGoogle Scholar
  2. Baum B.; Cherbas L. Drosophila cell lines as model systems and as an experimental tool. In: Dahmann C. (ed) Drosophila methods and protocols. Humana, Totowa, pp 391–424; 2008.Google Scholar
  3. Bourouis M.; Jarry B. Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance. EMBO J. 2: 1099–1104; 1983.PubMedGoogle Scholar
  4. Coonrod A.; Li F. Q.; Horwitz M. On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther. 4: 1313–1321; 1997.PubMedCrossRefGoogle Scholar
  5. Fraser M. J.; Ciszczon T.; Elick T.; Bauser C. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol. Biol. 5: 141–151; 1996.PubMedCrossRefGoogle Scholar
  6. Hammond A. M.; Bernstein E.; Beach D.; Hannon G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293–296; 2000.PubMedCrossRefGoogle Scholar
  7. Handler A. M. Use of the piggyBac transposon for germ-line transformation of insects. Insect Brioche Mol. Biol. 32: 1211–1220; 2002.CrossRefGoogle Scholar
  8. Handler A. M.; Harrell II R. A. Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol. Biol. 8: 449–457; 1999.PubMedCrossRefGoogle Scholar
  9. Lau N. C.; Robine N.; Martin R.; Chung W.-J.; Niki Y.; Berezikov E.; Kingston R.; Lai E. C. Abundant primary piRNAs, endo-siRNAs and microRNAs in a Drosophila ovary cell line. Genome Res. 19: 1776–1785; 2009.PubMedCrossRefGoogle Scholar
  10. Li X.; Heinrich J. C.; Scott M. J. piggyBac-mediated transposition in Drosophila melanogaster: an evaluation of the use of constitutive promoters to control transposase gene expression. Insect Mol. Biol. 10: 447–455; 2001.PubMedGoogle Scholar
  11. Niki Y.; Ymaguchi T.; Mahowald A. P. Establishment of stable cell lines of Drosophila germ-line stem cells. Proc. Natl. Acad. Sci. U.S.A. 103: 16325–16330; 2006.PubMedCrossRefGoogle Scholar
  12. Okamura K.; Ishizuka A.; Siomi H.; Siomi M. C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18: 1655–1666; 2004.PubMedCrossRefGoogle Scholar
  13. Schneider I. Cell line derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morph. 27: 353–365; 1972.PubMedGoogle Scholar
  14. Sumitani M.; Yamamoto D. S.; Oishi K.; Lee J. M.; Hatakeyama M. Germline transformation of the saefly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector. Insect Brioche Mol. Biol. 33: 449–458; 2003.CrossRefGoogle Scholar
  15. Tamura T.; Thlbert C.; Royer C.; Kanda T.; Abraham E.; Kamba M.; Komoto N.; Thomas J. L.; Mauchamp B.; Chavancy G.; Shirk P.; Fraser M.; Prudhomme J. C.; Couble P. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotech. 18: 81–84; 1999.Google Scholar

Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

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

Personalised recommendations