Skip to main content

Transient Transfection of Schneider Cells in the Study of Transcription Factors

  • Protocol
Transcription Factor Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 130))

Abstract

Transfections into Schneider’s Drosophila line 2 (abbreviated SL2 or S2) derived from Drosophila embryos (1) have been used to analyze activation properties of mammalian transcription factors (Table 1), to identify activation and inhibitory domains, as well as to investigate specific protein-protein interactions in vivo. SL2 cells are particularly suited to this task because they are devoid of many ubiquitous mammalian transcription factor activities and thus their transcriptional properties can be investigated in the absence of interference by endogenous factors. The usage of SL2 cells, as a host for studying the structure and function of heterologous, mammalian transcription factors, was originally reported by the group of R. Tjian to identify functional domains of AP-2 (2), Sp1 (3,4) and CTF/NF-I (5).

Table 1 Analysis of Heterologous Transcription Factors in SL2 Cells a

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (Canada)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (Canada)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (Canada)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (Canada)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schneider, I. (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–365.

    PubMed  CAS  Google Scholar 

  2. Williams, T., Admon, A., Luscher, B., and Tjian, R. (1988) Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev. 2, 1557–1569.

    Article  PubMed  CAS  Google Scholar 

  3. Courey, A. J. and Tjian, R. (1988) Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55, 887–898.

    Article  PubMed  CAS  Google Scholar 

  4. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 59, 827–836.

    Article  PubMed  CAS  Google Scholar 

  5. Mermod, N., O’Neill, E. A., Kelly, T. J., and Tjian, R. (1989) The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell 58, 741–753.

    Article  PubMed  CAS  Google Scholar 

  6. DiNocera, P. P. and Dawid, I. B. (1983) Transient expression of genes introduced into cultured cells of Drosophila. Proc. Natl. Acad. Sci. USA 80, 7095–7098.

    Article  CAS  Google Scholar 

  7. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds. (1997) Current Protocols in Molecular Biology, Wiley, New York.

    Google Scholar 

  8. Andrews, N. C. and Faller, D. V. (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19, 2499.

    Article  PubMed  CAS  Google Scholar 

  9. Pascal, E. and Tjian, R. (1991) Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev. 5, 1646–1656.

    Article  PubMed  CAS  Google Scholar 

  10. Yoshinaga, S. K. and Yamamoto, K. R. (1991) Signaling and regulation by a mammalian glucocorticoid receptor in Drosophila cells. Mol. Endocrinol. 5, 844–853.

    Article  PubMed  CAS  Google Scholar 

  11. Gregory, R. C., Taxman, D. J., Seshasayee, D., Kensinger, M. H., Bieker, J. J., and Wojchowski, D. M. (1996) Functional interaction of GATA1 with erythroid Krüppel-like factor and Sp1 at defined erythroid promoters. Blood 87, 1793–1801.

    PubMed  CAS  Google Scholar 

  12. Merika, M. and Orkin, S. H. (1995) Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Krüppel family proteins Sp1 and EKLF. Mol. Cell. Biol. 15, 2437–2447.

    PubMed  CAS  Google Scholar 

  13. Galera, P., Musso, M., Ducy, P., and Karsenty, G. (1994) c-Krox, a transcriptional regulator of type I collagen gene expression, is preferentially expressed in skin. Proc. Natl. Acad. Sci. USA 91, 9372–9376.

    Article  PubMed  CAS  Google Scholar 

  14. Chavrier, P., Vesque, C., Galliot, B., Vigneron, M., Dollé, P., Duboule, D., and Charnay, P. (1990) The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1,4 gene. EMBO J. 9, 1209–1218.

    PubMed  CAS  Google Scholar 

  15. Hagen, G., Müller, S., Beato, M., and Suske, G. (1994) Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J. 13, 3843–3851.

    PubMed  CAS  Google Scholar 

  16. Hagen, G., Dennig, J., Preiβ, A., Beato, M., and Suske, G. (1995) Functional analyses of the transcription factor Sp4 reveal properties distinct from Sp1 and Sp3. J. Biol. Chem. 270, 24,989–24,994.

    Article  PubMed  CAS  Google Scholar 

  17. Kobayashi, A., Sogawa, K., and Fujii, K. Y. (1996) Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene. J. Biol. Chem. 271, 12,310–12,316.

    Article  PubMed  CAS  Google Scholar 

  18. Seto, E., Lewis, B., and Shenk, T. (1993) Interaction between transcription factors Sp1 and YY1. Nature 365, 462–464.

    Article  PubMed  CAS  Google Scholar 

  19. Xu, J., Thompson, K. L., Shephard, L. B., Hudson, L. G., and Gill, G. N. (1993) T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites. J. Biol. Chem. 268, 16,065–16,073.

    PubMed  CAS  Google Scholar 

  20. Nehls, M. C., Grapilon, M. L., and Brenner, D. A. (1992) NF-I/Sp1 switch elements regulate collagen alpha 1(I) gene expression. DNA Cell. Biol. 11, 443–452.

    Article  PubMed  CAS  Google Scholar 

  21. Fischer, K.-D., Haese, A., and Nowock, J. (1993) Cooperation of GATA-1 and Sp1 can result in synergistic transcriptional activation or interference. J. Biol. Chem. 268, 23,915–23,923.

    PubMed  CAS  Google Scholar 

  22. Gegonne, A., Bosselut, R., Bailly, R. A., and Ghysdael, J. (1993) Synergistic activation of the HTLV1 LTR ets-responsive region by transcription factors ets1 and Sp1. EMBO J. 12, 1169–1178.

    PubMed  CAS  Google Scholar 

  23. Chen, X., Farmer, G., Zhu, H., Prywes, R., and Prives, C. (1993) Cooperative DNA binding of p53 with TFIID (TBP): a possible mechanism for transcriptional activation. Genes Dev. 7, 1837–1849.

    Article  PubMed  CAS  Google Scholar 

  24. Kerr, L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. G., Zhou, Q., Berk, A. J., and Verma, I. M. (1993) Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-kappa B. Nature 365, 412–419.

    Article  PubMed  CAS  Google Scholar 

  25. Yieh, L., Sanchez, H. B., and Osborne, T. F. (1995) Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter. Proc. Natl. Acad. Sci. USA 92, 6102–6106.

    Article  PubMed  CAS  Google Scholar 

  26. Roulet, E., Armentero, M. T., Krey, G., Corthesy, B., Dreyer, C., Mermod, N., and Wahli, W. (1995) Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain. Mol. Cell. Biol. 15, 5552–5562.

    PubMed  CAS  Google Scholar 

  27. Lin, S.-Y., Black, A. R., Kostic, D., Pajovic, S., Hoover, C. N., and Azizkhan, J. C. (1996) Cell cycle-regulated association of E2F and Sp1 is related to their functional interaction. Mol. Cell. Biol. 16, 1668–1675.

    PubMed  CAS  Google Scholar 

  28. Lee, Y. H., Williams, S. C., Baer, M., Sterneck, E., Gonzalez, F. J., and Johnson, P. F. (1997) The ability of C/EBPβ but not C/EBPα to synergize with an Sp1 protein is specified by the leucine zipper and activation domain. Mol. Cell. Biol. 17, 2038–2047.

    PubMed  CAS  Google Scholar 

  29. Farmer, G., Friedlander, P., Colgan, J., Manley, J. L., and Prives, C. (1996) Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein. Nucl. Acids Res. 24, 4281–4288.

    Article  PubMed  CAS  Google Scholar 

  30. Trivedi, A., Vilalta, A., Gopalan, S., and Johnson, D. L. (1996) TATA-binding protein is limiting for both TATA-containing and TATA-lacking RNA polymerase III promoters in Drosophila cells. Mol. Cell. Biol. 16, 6909–6916.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2000 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Suske, G. (2000). Transient Transfection of Schneider Cells in the Study of Transcription Factors. In: Tymms, M.J. (eds) Transcription Factor Protocols. Methods in Molecular Biology™, vol 130. Humana Press. https://doi.org/10.1385/1-59259-686-X:175

Download citation

  • DOI: https://doi.org/10.1385/1-59259-686-X:175

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-573-7

  • Online ISBN: 978-1-59259-686-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics