Isolation of recombinant MVA using F13L selection

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 890; p. 93
• Main Authors: Sánchez-Puig, Juana M, Lorenzo, María M, Blasco, Rafael
• Format: Journal Article
• Language: English
• Published: United States 2012
• Subjects: Animals; Base Sequence; Cells, Cultured; Cloning, Molecular; Cricetinae; DNA, Recombinant; Gene Knockout Techniques; Genetic Markers; Membrane Proteins - genetics; Promoter Regions, Genetic; Transduction, Genetic; Vaccinia virus - genetics; Vaccinia virus - growth & development; Vaccinia virus - isolation & purification; Viral Envelope Proteins - genetics; Viral Load; Virus Cultivation
• ISSN: 1940-6029
• DOI: 10.1007/978-1-61779-876-4_5

Modified vaccinia virus Ankara (MVA) has become a widely used vector for vaccine and laboratory purposes. Despite significant advances in recombinant MVA technology, the isolation of recombinant viruses remains a tedious and difficult process. This chapter describes the use of an efficient and easy-to-use selection system adapted for MVA. The system is based on the requirement of the viral gene F13L for efficient virus spread in cell culture, which results in a severe block in virus transmission when F13L gene is deleted (Blasco R, Moss B. J Virol 65:5910-5920, 1991; Blasco R, Moss B. J Virol 66:4170-4179, 1992). The insertion of foreign genes in the MVA genome is accomplished by recombination of a transfected plasmid carrying the foreign genes and the F13L with the genome of an F13L knockout virus. Subsequently, selection of virus recombinants is carried out by serial passage and/or plaque purification of viruses that have recovered the F13L gene.