Isolation of recombinant MVA using F13L selection.
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.
Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain.
Juana M
JM
Sánchez-Puig
María M
MM
Lorenzo
Rafael
R
Blasco
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D004274:DNA, Recombinant; D055786:Gene Knockout Techniques; D005819:Genetic Markers; D008565:Membrane Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D014616:Vaccinia virus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D014759:Viral Envelope Proteins / Q000235:genetics*; D019562:Viral Load; D014776:Virus Cultivation
D004274:DNA, Recombinant; D005819:Genetic Markers; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; C084759:p37 protein, Vaccinia virus