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

  • Manon Gaudreault
  • Danielle Larouche
  • Lucie Germain
  • Sylvain L. Guérin
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 989)

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.

Key words

Stem cells Epidermis Cornea Corneal epithelial cells Tissue engineering Transcription factor Sp1 EMSA Gel shift Mobility shift assay Western blot 

Notes

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.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Manon Gaudreault
    • 1
  • Danielle Larouche
    • 2
    • 3
  • Lucie Germain
    • 4
    • 5
    • 6
  • Sylvain L. Guérin
    • 7
    • 6
  1. 1.LOEX/CUO-Recherche QuébecQuébecCanada
  2. 2.Génie tissulaire et régénération: LOEX—Centre de recherche FRSQ du Centre hospitalier affilié universitaire de QuébecQuébecCanada
  3. 3.LOEX de l’Université LavalQuebecCanada
  4. 4.Génie tissulaire et régénération, LOEX—Centre de recherche FRSQ du Centre hospitalier affilié universitaire de QuébecQuébecCanada
  5. 5.Centre LOEX de l’Université LavalQuébecCanada
  6. 6.Département d’ophtalmologie, Faculté de médecineUniversité LavalQuébecCanada
  7. 7.LOEX/CUO-Recherche, Génie tissulaire et régénération : LOEX—Centre de recherche FRSQ du Centre hospitalier affilié universitaire de QuébecQuebecCanada

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