Abstract
Electrophoretic mobility shift assay and Western blot are simple, efficient, and rapid methods for the study of DNA–protein interactions and expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem-cells through the culture process is essential to produce high quality substitutes. However as such cells are passaged in culture, they often lose their ability to proliferate, a process likely to be determined by the altered expression of nuclear-located transcription factors such as Sp1, whose expression has been documented to be required for cell adhesion, migration, and differentiation. Our recent studies demonstrated that reconstructed tissues exhibiting poor histological and structural characteristics are also those that were produced with epithelial cells in which expression and DNA binding of Sp1 was reduced in vitro. Therefore, monitoring both the expression and DNA binding of this transcription factor in human skin and corneal epithelial cells might prove a particularly useful tool for selecting which cells are to be used for tissue reconstruction.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Green H, Rheinwald JG, Sun TT (1977) Properties of an epithelial cell type in culture: the epidermal keratinocyte and its dependence on products of the fibroblast. Prog Clin Biol Res 17:493–500
McLoughlin CB (1961) The importance of mesenchymal factors in the differentiation of chick epidermis. J Embryol Exp Morphol 9:370–384
Melbye SW, Karasek MA (1973) Some characteristics of a factor stimulating skin epithelial cell growth in vitro. Exp Cell Res 79:279–286
Wessells NK (1964) Substrate and nutrient effects upon epidermal basal cell orientation and proliferation. Proc Natl Acad Sci U S A 52:252–259
Masson-Gadais B, Fugere C, Paquet C, Leclerc S, Lefort NR, Germain L, Guerin SL (2006) The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3. J Cell Physiol 206:831–842
Gaudreault M, Carrier P, Larouche K, Leclerc S, Giasson M, Germain L, Guerin SL (2003) Influence of sp1/sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues. Invest Ophthalmol Vis Sci 44:1447–1457
Larouche D, Paquet C, Fradette J, Carrier P, Auger FA, Germain L (2009) Regeneration of skin and cornea by tissue engineering. Methods Mol Biol 482:233–256
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Li L, Davie JR (2010) The role of Sp1 and Sp3 in normal and cancer cell biology. Ann Anat 192:275–283
Zhao C, Meng A (2005) Sp1-like transcription factors are regulators of embryonic development in vertebrates. Dev Growth Differ 47:201–211
Li L, He S, Sun JM, Davie JR (2004) Gene regulation by Sp1 and Sp3. Biochem Cell Biol 82:460–471
Leclerc S, Eskild W, Guerin SL (1997) The rat growth hormone and human cellular retinol binding protein 1 genes share homologous NF1-like binding sites that exert either positive or negative influences on gene expression in vitro. DNA Cell Biol 16:951–967
Roder K, Schweizer M (2001) Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA). Biotechnol Appl Biochem 33:209–214
Gaudreault M, Vigneault F, Leclerc S, Guerin SL (2007) Laminin reduces expression of the human alpha6 integrin subunit gene by altering the level of the transcription factors Sp1 and Sp3. Invest Ophthalmol Vis Sci 48:3490–3505
Vossen KM, Fried MG (1997) Sequestration stabilizes lac repressor-DNA complexes during gel electrophoresis. Anal Biochem 245:85–92
Acknowledgments
The authors would like to thank current and former members of the LOEX and LOEX/CUO-Recherche laboratories who have contributed to develop the foregoing protocols.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Gaudreault, M., Larouche, D., Germain, L., Guérin, S.L. (2013). Qualitatively Monitoring Binding and Expression of the Transcription Factor Sp1 as a Useful Tool to Evaluate the Reliability of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction. In: Turksen, K. (eds) Skin Stem Cells. Methods in Molecular Biology, vol 989. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-330-5_11
Download citation
DOI: https://doi.org/10.1007/978-1-62703-330-5_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-329-9
Online ISBN: 978-1-62703-330-5
eBook Packages: Springer Protocols