Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA
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.
Key wordsRecombinant poliovirus cDNA cloning Restriction cloning Bacterial transformation In vitro transcription RNA transfection Virus recovery
- 2.Burrill CP et al (2013) Poliovirus: generation and characterisation of mutants. Curr Protoc Microbiol. Chapter 15:Unit 15H.2