CRISPR/Cas9 Editing for Gaucher Disease Modelling
Abstract
:1. Introduction
2. Results
2.1. Monocytic GBA1 Mutant Cells: A Promising Cellular Model of GD
2.2. U87 GBA1 Mutant Cells: A Useful Tool to Study Neuroinflammation in GD
2.2.1. Endoplasmic Reticulum Associated Degradation (ERAD) and ER Stress
2.2.2. Inflammatory Markers
2.2.3. Accumulation of α-Synuclein and Cell Death
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. MG132 Treatment
4.3. CRISPR/Cas9 GBA1 Editing
4.4. Off-Target Analysis
4.5. DNA Extraction, GBA1 Exon 3 Amplification and Sequencing
4.6. Protein Extraction and Western Blot
4.7. Endo-H and Endo-F Digestion
4.8. Enzymatic Activity
4.9. RNA Extraction, Reverse Transcription and Quantitative Real Time PCR
4.10. LysoGL1 Measurement
4.11. Cytokine Measurement
4.12. Annexin-V Staining
4.13. Lactate Dehydrogenase (LDH) Activity
4.14. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Endo-F | Endoglycase F |
Endo-H | Endoglycase H |
ER | Endoplasmic reticulum |
ERAD | ER associated degradation |
GCase | acid β-glucosidase |
GD | Gaucher disease |
GlcCer | Glucosylceramide |
HEK | Human Embryonic Kidney cells 293 |
IL-1β | Interleukin-1β |
IL-6 | Interleukin-6 |
iPSC | Induced pluripotent stem cells |
LDH | Lactate dehydrogenase |
LysoGL1 | Glucosylsphingosine |
MW | Molecular weight |
nGD | Neuronopathic GD |
PD | Parkinson’s disease |
RNAi | RNA interference |
THP-1 | Human monocytic cell line deriving from an acute monocytic leukemia patient |
TNFα | Tumor necrosis factor α |
U87 | Glioblastoma U87 cell line |
UPR | Unfolded Protein Response |
UPS | Ubiquitin-Proteasomal System |
WB | Western Blot |
α-syn | α-synuclein |
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Pavan, E.; Ormazabal, M.; Peruzzo, P.; Vaena, E.; Rozenfeld, P.; Dardis, A. CRISPR/Cas9 Editing for Gaucher Disease Modelling. Int. J. Mol. Sci. 2020, 21, 3268. https://doi.org/10.3390/ijms21093268
Pavan E, Ormazabal M, Peruzzo P, Vaena E, Rozenfeld P, Dardis A. CRISPR/Cas9 Editing for Gaucher Disease Modelling. International Journal of Molecular Sciences. 2020; 21(9):3268. https://doi.org/10.3390/ijms21093268
Chicago/Turabian StylePavan, Eleonora, Maximiliano Ormazabal, Paolo Peruzzo, Emilio Vaena, Paula Rozenfeld, and Andrea Dardis. 2020. "CRISPR/Cas9 Editing for Gaucher Disease Modelling" International Journal of Molecular Sciences 21, no. 9: 3268. https://doi.org/10.3390/ijms21093268