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Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction

  • Gaëtan Le-Bel
  • Sergio Cortez Ghio
  • Danielle Larouche
  • Lucie Germain
  • Sylvain L. Guérin
Protocol
Part of the Methods in Molecular Biology book series

Abstract

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA–protein interactions and protein 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, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

Keywords

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

Abbreviations

AEBSF

4-(2-Aminoethyl)benzenesulfonyl fluoride

anhCEC

Adult normal human corneal epithelial cells

anhK

Adult normal human keratinocytes

APS

Ammonium persulfate

ATP

Adenosine triphosphate

BCA

Bicinchoninic acid

ccDME-Ham

Complete corneal epithelial cell culture medium

ckDME-Ham

Complete keratinocyte culture medium

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

DNA

Deoxyribonucleic acid

DTT

Dithiothreitol

ECL

Enhanced chemiluminescence

EDTA

Ethylenediaminetetraacetic acid

EGF

Epidermal growth factor

EMSA

Electrophoretic mobility shift assay

Ham

Ham’s medium

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

i3T3

Irradiated Swiss 3T3

iHFL

Irradiated human feed layer

MMP9

Matrix metallopeptidase 9

NE

Nuclear extract

NFI

Nuclear factor I

nhCEC

Normal human corneal epithelial cell

nhK

Normal human keratinocytes

nnhCEC

Newborn normal human corneal epithelial cells

nnhK

Newborn normal human keratinocytes

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline

PMSF

Phenylmethylsulfonyl fluoride

PNK

T4 polynucleotide kinase

poly(dI:dC)

Poly(deoxyinosinic-deoxycytidylic) acid sodium salt

PVDF

Polyvinylidene fluoride

SCC

Supershifted complexes

SDS

Sodium dodecyl sulfate

Sp1

Specificity protein 1

STE

Sodium Tris–HCl EDTA

TBS

Tris Buffered Saline

TBS-T

Tris Buffered Saline tween

tDMEM

Tissue transport medium

TE

Tris–HCl EDTA

TEMED

Tetramethylethylenediamine

TM

Melting temperature

Notes

Acknowledgments

The authors would like to thank current and former members of the LOEX and CUO-Recherche laboratories who contributed to develop and improve the foregoing protocols.

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Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Gaëtan Le-Bel
    • 1
    • 2
    • 3
    • 4
  • Sergio Cortez Ghio
    • 2
    • 3
  • Danielle Larouche
    • 2
    • 3
  • Lucie Germain
    • 1
    • 2
    • 3
    • 4
  • Sylvain L. Guérin
    • 1
    • 4
  1. 1.Centre Universitaire d’Ophtalmologie—Recherche (CUO Recherche), CHU de QuébecQuébec (Qc)Canada
  2. 2.Centre de recherche en organogénèse expérimentale de l’Université Laval/LOEX, CHU de Québec—Université Laval Research CenterQuébec (Qc)Canada
  3. 3.Department of Surgery, Faculty of MedicineUniversité LavalQuébec (Qc)Canada
  4. 4.Department of Ophthalmology, Faculty of MedicineUniversité LavalQuébec (Qc)Canada

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