Development of a FLP/frt System for Generating Helper-Dependent Adenoviral Vectors
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Résumé
Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences have a large cloning capacity and have been reported to provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of generating HD vectors involves co-infecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated excision of the packaging signal renders the helper virus genome unpackageable but still able to replicate and to provide helper functions for HD vector propagation. HD vector titer is increased by serial co-infections. Typically, helper virus contamination is ≤1% pre- and ≤0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. Alternative methods of selection against the helper virus may achieve this goal, especially when combined with Cre/loxP. We describe the development of a system for generating HD vectors based on site-specific recombination between frt sites catalyzed by FLP recombinase and show by direct comparison that the FLP/frt and Cre/loxP systems are equivalent with respect to HD vector amplification efficiency and helper virus contamination levels. Availability of a second recombinase system for HD vector production will enhance the utility and flexibility of HD vectors. Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences have a large cloning capacity and have been reported to provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of generating HD vectors involves co-infecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated excision of the packaging signal renders the helper virus genome unpackageable but still able to replicate and to provide helper functions for HD vector propagation. HD vector titer is increased by serial co-infections. Typically, helper virus contamination is ≤1% pre- and ≤0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. Alternative methods of selection against the helper virus may achieve this goal, especially when combined with Cre/loxP. We describe the development of a system for generating HD vectors based on site-specific recombination between frt sites catalyzed by FLP recombinase and show by direct comparison that the FLP/frt and Cre/loxP systems are equivalent with respect to HD vector amplification efficiency and helper virus contamination levels. Availability of a second recombinase system for HD vector production will enhance the utility and flexibility of HD vectors.
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Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découle