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Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14759-63.

Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome.

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  • 1Max von Pettenkofer-Institut für Hygiene und Mikrobiologie, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany.


A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.

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