Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination.
The large capacity of vaccinia virus (VAC) for added DNA, cytoplasmic expression and broad host range make it a popular choice for gene delivery, despite the burdensome need for multiple plaque purifications to isolate recombinants. Here we describe how a bacterial artificial chromosome (BAC) containing the entire VAC genome can be engineered in Escherichia coli by homologous recombination using bacteriophage lambda-encoded enzymes. The engineered VAC genomes can then be used to produce clonally pure recombinant viruses in mammalian cells without the need for plaque purification.
Domi
Arban
A
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Bethesda, Maryland 20892-0445, USA.
Moss
Bernard
B
D010582:Bacteriophage lambda / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D016679:Genome, Viral; D011995:Recombination, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014169:Transformation, Bacterial / Q000235:genetics; D014615:Vaccinia / Q000235:genetics*