Skip to main content

Using siRNA Knockdown in HaCaT Cells to Study Transcriptional Control of Epidermal Proliferation Potential

  • Protocol
  • First Online:
Epidermal Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 585))

Abstract

Compared to primary keratinocytes, HaCaT cells are easier to transfect and yet still maintain at least some features of normal epidermal proliferation and differentiation. This chapter describes methods used in our laboratory to maintain HaCaT cells in an undifferentiated state and to use the siRNA technology to efficiently deplete a gene product of interest from these cells. We also provide protocols on how to couple siRNA knockdown with a clonal assay to examine keratinocyte proliferation potential and a luciferase reporter assay to examine promoter regulation.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • 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

Similar content being viewed by others

References

  1. Potten, C. S., and Morris, R. J. (1988) Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45–62.

    PubMed  CAS  Google Scholar 

  2. Lavker, R. M., and Sun, T. T. (2000) Epidermal stem cells: properties, markers, and location. Proc. Natl. Acad. Sci. USA 97, 13473–13475.

    Article  PubMed  CAS  Google Scholar 

  3. Jones, P. H., and Watt, F. M. (1993) Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73, 713–724.

    Article  PubMed  CAS  Google Scholar 

  4. Jones, P. H., Harper, S., and Watt, F. M. (1995) Stem cell patterning and fate in human epidermis. Cell 80, 83–93.

    Article  PubMed  CAS  Google Scholar 

  5. Watt, F. M. (2001) Stem cell fate and patterning in mammalian epidermis. Curr. Opin. Genet. Dev. 11, 410–417.

    Article  PubMed  CAS  Google Scholar 

  6. Barrandon, Y., and Green, H. (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84, 2302–2306.

    Article  PubMed  CAS  Google Scholar 

  7. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771.

    Article  PubMed  CAS  Google Scholar 

  8. Schoop, V. M., Mirancea, N., and Fusenig, N. E. (1999) Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts. J. Invest. Dermatol. 112, 343–353.

    Article  PubMed  CAS  Google Scholar 

  9. Wan, H., Yuan, M., Simpson, C., Allen, K., Gavins, F. N., Ikram, M. S., et al. (2007) Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines. Stem Cells 25, 1286–1297.

    Article  PubMed  CAS  Google Scholar 

  10. Wan, H., Stone, M. G., Simpson, C., Reynolds, L. E., Marshall, J. F., Hart, I. R., et al. (2003) Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes. J. Cell Sci. 116, 4239–4248.

    Article  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

© 2010 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Wells, J., Dai, X. (2010). Using siRNA Knockdown in HaCaT Cells to Study Transcriptional Control of Epidermal Proliferation Potential. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 585. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-380-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-380-0_9

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-379-4

  • Online ISBN: 978-1-60761-380-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics