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Molecular genetics of herpesviruses: a recombinant technology approach.
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Herpesvirus genetics have long been hindered by the large size of the typical herpesvirus genome and the consequent recalcitrance of these genomes to manipulation by standard molecular genetics techniques. However, two primary strategies have emerged that allow for the generation of targeted viral mutants. With these mutants, investigators can pursue critical questions regarding the relationship between specific viral genetic elements and the viral life cycle. The first strategy of viral genome manipulation utilizes the mammalian homologous recombination machinery to introduce specific changes into the native viral genome. This approach involves construction of a targeting vector containing both the desired mutation and a significant flanking viral sequence to permit efficient recombination. The targeting vector is then introduced into mammalian cells, along with viral DNA, and recombinant virus is subsequently selected, harvested, and purified. The second, and more recent, approach utilizes bacterial artificial chromosome (BAC) technology to reconstitute complete herpesvirus genomes in the context of a prokaryote, E. coli. This artificial genome is then manipulated in, and purified from, E. coli before introduction into a mammalian background in which viral phenotypes can be assessed. Both strategies are discussed in this review, with particular emphasis on the homologous recombination strategies that have continued to be a powerful genetic tool in many herpesvirus systems.