Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome.
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.
Messerle
M
M
Max von Pettenkofer-Institut für Hygiene und Mikrobiologie, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. Messerle@lmb.uni-muenchen.de
Crnkovic
I
I
Hammerschmidt
W
W
Ziegler
H
H
Koszinowski
U H
UH
D000818:Animals; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral; D006564:Herpesviridae / Q000235:genetics*; D051379:Mice; D016296:Mutagenesis