Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method
Abstract
:1. Introduction
2. Materials and Methods
2.1. rAAV Vector Production in HEK293 Cells
2.2. rAAV Vector Production in Sf9 Cells
2.2.1. Plasmid Constructs
2.2.2. Generation and Production of Recombinant Baculoviral Stocks
2.3. Cell Culture
2.4. Neonatal Rat Ventricular Myocytes (NRVMs)
2.5. Human Induced-Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
2.6. Flow Cytometry
2.7. Western Blot Analysis for VP Proteins
2.8. AAV Vector Performance in Mice
2.9. Genomic DNA Extraction and Vector Genome Copy Number Analysis
2.10. RNA Extraction and GFP Expression Via qPCR
3. Results and Discussion
3.1. Comparison of rAAV Produced by HEK293 and BEV Systems
3.2. Characterisation of Capsid Proteins for the Various rAAV Serotypes
3.3. Comparison of rAAV6-GFP Transduction Efficiency in Cardiomyocytes
3.4. Comparison of rAAV6-GFP Transduction Efficiency In Vivo
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Rao, R.; Farraha, M.; Logan, G.J.; Igoor, S.; Kok, C.Y.; Chong, J.J.H.; Alexander, I.E.; Kizana, E. Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method. Viruses 2022, 14, 1623. https://doi.org/10.3390/v14081623
Rao R, Farraha M, Logan GJ, Igoor S, Kok CY, Chong JJH, Alexander IE, Kizana E. Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method. Viruses. 2022; 14(8):1623. https://doi.org/10.3390/v14081623
Chicago/Turabian StyleRao, Renuka, Melad Farraha, Grant J. Logan, Sindhu Igoor, Cindy Y. Kok, James J. H. Chong, Ian E. Alexander, and Eddy Kizana. 2022. "Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method" Viruses 14, no. 8: 1623. https://doi.org/10.3390/v14081623
APA StyleRao, R., Farraha, M., Logan, G. J., Igoor, S., Kok, C. Y., Chong, J. J. H., Alexander, I. E., & Kizana, E. (2022). Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method. Viruses, 14(8), 1623. https://doi.org/10.3390/v14081623