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Due to the large genome size and the slow replication kinetics <jats:italic>in vitro<\/jats:italic> of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in <jats:italic>E. coli<\/jats:italic>. This technique allows the maintenance of viral genomes as a plasmid in <jats:italic>E. coli<\/jats:italic> and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in <jats:italic>E. coli<\/jats:italic> using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV\u201068), a mouse model for gammaherpesvirus infections, as an example. 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