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