Abstract
The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.
The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5′ noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.
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References
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Bujaki, E. (2016). Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA. 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_12
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DOI: https://doi.org/10.1007/978-1-4939-3292-4_12
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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