5′RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa
Abstract
:1. Introduction
2. Results
2.1. Selection of Highly Functional RTMs Using a Fluorescence-Based Screening System
2.2. Endogenous 5′ Trans-Splicing in Keratinocytes
2.3. Endogenous Correction of COL7A1 in RDEB Patient Cells
2.4. C7 Expression in Skin Equivalents
3. Discussion
4. Materials and Methods
4.1. Cloning of Screening Constructs
4.2. Plasmid Transfection and Trans-Splicing Evaluation
4.3. RNA Isolation
4.4. cDNA Amplification and PCR
4.5. Cloning of Constructs for Endogenous Experiments
4.6. Viral Transduction
4.7. Semi-Quantitative (sq)RT-PCR
4.8. Western Blot Analysis
4.9. Immunofluorescence Staining of Type VII Collagen in Keratinocytes
4.10. Generation of Skin Equivalents
4.11. Immunofluorescence Staining of Type VII Collagen and Laminin 332 in Skin Equivalents
4.12. Cells
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mayr, E.; Ablinger, M.; Lettner, T.; Murauer, E.M.; Guttmann-Gruber, C.; Piñón Hofbauer, J.; Hainzl, S.; Kaiser, M.; Klausegger, A.; Bauer, J.W.; et al. 5′RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa. Int. J. Mol. Sci. 2022, 23, 1732. https://doi.org/10.3390/ijms23031732
Mayr E, Ablinger M, Lettner T, Murauer EM, Guttmann-Gruber C, Piñón Hofbauer J, Hainzl S, Kaiser M, Klausegger A, Bauer JW, et al. 5′RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa. International Journal of Molecular Sciences. 2022; 23(3):1732. https://doi.org/10.3390/ijms23031732
Chicago/Turabian StyleMayr, Elisabeth, Michael Ablinger, Thomas Lettner, Eva M. Murauer, Christina Guttmann-Gruber, Josefina Piñón Hofbauer, Stefan Hainzl, Manfred Kaiser, Alfred Klausegger, Johann W. Bauer, and et al. 2022. "5′RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa" International Journal of Molecular Sciences 23, no. 3: 1732. https://doi.org/10.3390/ijms23031732