Abstract
The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing the phage T7 RNA polymerase. This method permits the rapid recovery of modified viruses that can be subsequently amplified on adequate cell-lines.
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Acknowledgments
This work was supported by the Transverse Research Program PTR-276, the Agence Nationale pour la Recherche (ANR 09 MIEN 019), the Fondation pour la Recherche Médicale (FRM DMI20091117313).
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Bessaud, M., Pelletier, I., Blondel, B., Delpeyroux, F. (2016). A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses. In: Martín, J. (eds) Poliovirus. Methods in Molecular Biology, vol 1387. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3292-4_13
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DOI: https://doi.org/10.1007/978-1-4939-3292-4_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3291-7
Online ISBN: 978-1-4939-3292-4
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