A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Abstract Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it de...

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Autores principales: V. Kalidasan, Wai Hoe Ng, Oluwaseun Ayodeji Ishola, Nithya Ravichantar, Jun Jie Tan, Kumitaa Theva Das
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Publicado: Nature Portfolio 2021
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Acceso en línea:https://doaj.org/article/2f0a52a5611d43b2989a84073a2f6158
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spelling oai:doaj.org-article:2f0a52a5611d43b2989a84073a2f61582021-12-02T18:51:28ZA guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system10.1038/s41598-021-98657-72045-2322https://doaj.org/article/2f0a52a5611d43b2989a84073a2f61582021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-98657-7https://doaj.org/toc/2045-2322Abstract Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.V. KalidasanWai Hoe NgOluwaseun Ayodeji IsholaNithya RavichantarJun Jie TanKumitaa Theva DasNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
V. Kalidasan
Wai Hoe Ng
Oluwaseun Ayodeji Ishola
Nithya Ravichantar
Jun Jie Tan
Kumitaa Theva Das
A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
description Abstract Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.
format article
author V. Kalidasan
Wai Hoe Ng
Oluwaseun Ayodeji Ishola
Nithya Ravichantar
Jun Jie Tan
Kumitaa Theva Das
author_facet V. Kalidasan
Wai Hoe Ng
Oluwaseun Ayodeji Ishola
Nithya Ravichantar
Jun Jie Tan
Kumitaa Theva Das
author_sort V. Kalidasan
title A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
title_short A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
title_full A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
title_fullStr A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
title_full_unstemmed A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
title_sort guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
publisher Nature Portfolio
publishDate 2021
url https://doaj.org/article/2f0a52a5611d43b2989a84073a2f6158
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