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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Potten, C. S., and Morris, R. J. (1988) Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45–62.
Lavker, R. M., and Sun, T. T. (2000) Epidermal stem cells: properties, markers, and location. Proc. Natl. Acad. Sci. USA 97, 13473–13475.
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.
Jones, P. H., Harper, S., and Watt, F. M. (1995) Stem cell patterning and fate in human epidermis. Cell 80, 83–93.
Watt, F. M. (2001) Stem cell fate and patterning in mammalian epidermis. Curr. Opin. Genet. Dev. 11, 410–417.
Barrandon, Y., and Green, H. (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84, 2302–2306.
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.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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