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

• DOI: 10.1073/pnas.94.26.14759
• Publication Date: 2002-07-26T14:31:39Z
• Authors: Martin Messerle; Irena Crnkovic; Wolfgang Hammersc...; Heike Ziegler; Ulrich H. Koszino...
• Keywords: Mice; 0303 health sciences; 03 medical and health sciences; Mutagenesis; Escherichia coli; Animals; Genome, Viral; Chromosomes, Bacterial; Cloning, Molecular; Herpesviridae; 3. Good health

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.