| Download | - View final version: Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning (PDF, 3.9 MiB)
- View supplementary information: Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning (PDF, 396 KiB)
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| DOI | Resolve DOI: https://doi.org/10.1016/j.omtm.2023.02.013 |
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| Author | Search for: Broussau, Sophie1; Search for: Lytvyn, Viktoria1; Search for: Simoneau, Mélanie1; Search for: Guilbault, Claire1; Search for: Leclerc, Mélanie1; Search for: Nazemi-Moghaddam, Nazila1; Search for: Coulombe, Nathalie1; Search for: Elahi, Seyyed Mehdy1; Search for: McComb, Scott1ORCID identifier: https://orcid.org/0000-0002-4616-7864; Search for: Gilbert, Rénald1 |
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| Affiliation | - National Research Council of Canada. Human Health Therapeutics
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| Funder | Search for: National Research Council Canada |
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| Format | Text, Article |
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| Subject | lentivirus; packaging cells; cloning; suspension culture; serum-free; scale-up; gene induction; lentiviral vector; HEK293; cell therapy |
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| Abstract | Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method’s effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3 × 10⁸ transduction units (TU)/mL in the culture medium under batch mode without concentration. |
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| Publication date | 2023-02-26 |
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| Publisher | Elsevier
The American Society of Gene and Cell Therapy |
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| Licence | |
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| In | |
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| Language | English |
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| Peer reviewed | Yes |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 0ab78c3e-3b74-4dba-8bf9-39413f3d7964 |
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| Record created | 2023-04-11 |
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| Record modified | 2023-04-18 |
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