Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Generation of Split GFP Expressing Stable Cells
2.3. CRISPR Editing
2.4. DNA and RNA Validation of CRISPR Editing
2.5. In-Gel Detection of NLuc Activity
2.6. Proteomic Validation of CRISPR Editing
2.7. Quantitation of Absolute Collagen Levels
2.8. Hydroxyproline Assay
2.9. Bioluminescence Imaging
3. Results
3.1. CRISPR-Cas9 Editing of Col1a2
3.2. Quantitation of NLuc-PC-I
3.3. Direct Imaging and Quantitation of NLuc-PC-I in Cells
3.4. Circadian Fluctuations of Procollagen-I
3.5. NLuc-PC-I Response to Known Collagen Modulators
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Calverley, B.C.; Kadler, K.E.; Pickard, A. Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging. Cells 2020, 9, 2070. https://doi.org/10.3390/cells9092070
Calverley BC, Kadler KE, Pickard A. Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging. Cells. 2020; 9(9):2070. https://doi.org/10.3390/cells9092070
Chicago/Turabian StyleCalverley, Ben C., Karl E. Kadler, and Adam Pickard. 2020. "Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging" Cells 9, no. 9: 2070. https://doi.org/10.3390/cells9092070