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Biotechnology Letters

, Volume 33, Issue 12, pp 2361–2366 | Cite as

Inducible protein expression in Drosophila Schneider 2 cells using the lac operator–repressor system

  • Motoaki Wakiyama
  • Reiko Muramatsu
  • Yoko Kaitsu
  • Mariko Ikeda
  • Shigeyuki Yokoyama
Original Research Paper

Abstract

Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO4 or CdCl2 as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator–repressor regulatory system functioned in chromosomally integrated cell lines.

Keywords

Drosophila S2 cells lac operator–repressor Inducible expression IPTG 

Notes

Acknowledgments

This work was supported by the Targeted Proteins Research Program (TPRP), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

References

  1. Angelichio ML, Beck JA, Johansen H, Ivey-Hoyle M (1991) Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells. Nucleic Acids Res 19:5037–5043PubMedCrossRefGoogle Scholar
  2. Bahl CP, Wu R, Itakura K, Katagiri N, Narang SA (1976) Chemical and enzymatic synthesis of lactose operator of Escherichia coli and its binding to lactose repressor. Proc Natl Acad Sci USA 73:91–94PubMedCrossRefGoogle Scholar
  3. Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16:1043–1061PubMedCrossRefGoogle Scholar
  4. Cherbas L, Cherbas P (2007) Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression. Methods Mol Biol 388:317–340PubMedCrossRefGoogle Scholar
  5. Cherbas L, Moss R, Cherbas P (1994) Transformation techniques for Drosophila cell lines. Methods Cell Biol 44:161–179PubMedCrossRefGoogle Scholar
  6. Chung YT, Keller EB (1990) Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter. Mol Cell Biol 10:6172–6180PubMedGoogle Scholar
  7. Cronin CA, Gluba W, Scrable H (2001) The lac operator-repressor system is functional in the mouse. Genes Dev 15:1506–1517PubMedCrossRefGoogle Scholar
  8. Figge J, Wright C, Collins CJ, Roberts TM, Livingston DM (1988) Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells. Cell 52:713–722PubMedCrossRefGoogle Scholar
  9. Hu MC, Davidson N (1987) The inducible lac operator-repressor system is functional in mammalian cells. Cell 48:555–566PubMedCrossRefGoogle Scholar
  10. Johansen H, van der Straten A, Sweet R, Otto E, Maroni G, Rosenberg M (1989) Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells. Genes Dev 3:882–889PubMedCrossRefGoogle Scholar
  11. Mukai T, Wakiyama M, Sakamoto K, Yokoyama S (2010) Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells. Protein Sci 19:440–448PubMedGoogle Scholar
  12. Takimoto K, Wakiyama M, Yokoyama S (2009) Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression. RNA 15:1078–1089PubMedCrossRefGoogle Scholar
  13. Wakiyama M, Matsumoto T, Yokoyama S (2005) Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells. Biochem Biophys Res Commun 331:1163–1170PubMedCrossRefGoogle Scholar
  14. Wakiyama M, Kaitsu Y, Yokoyama S (2006) Cell-free translation system from Drosophila S2 cells that recapitulates RNAi. Biochem Biophys Res Commun 343:1067–1071PubMedCrossRefGoogle Scholar
  15. Yannoni YM, White K (1999) Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV. J Cell Sci 112:4501–4512PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Motoaki Wakiyama
    • 1
  • Reiko Muramatsu
    • 1
  • Yoko Kaitsu
    • 1
  • Mariko Ikeda
    • 1
  • Shigeyuki Yokoyama
    • 1
    • 2
  1. 1.RIKEN Systems and Structural Biology CenterYokohamaJapan
  2. 2.The University of Tokyo, Graduate School of ScienceTokyoJapan

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