A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa.

Autor: Zwicklhuber J; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Kocher T; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Liemberger B; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Hainzl S; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Bischof J; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Strunk D; Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University, 5020 Salzburg, Austria., Raninger AM; Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University, 5020 Salzburg, Austria., Gratz I; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria.; Department of Biosciences and Medical Biology, University of Salzburg, 5020 Salzburg, Austria., Wally V; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Guttmann-Gruber C; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Hofbauer JP; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Bauer JW; Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria., Koller U; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2023 Mar 08; Vol. 24 (6). Date of Electronic Publication: 2023 Mar 08.
DOI: 10.3390/ijms24065197
Abstrakt: Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17 mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP- COL17A1 mut -expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.
Databáze: MEDLINE
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