Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors

Abstract

Homologous recombination has been the exclusive means of introducing foreign DNA into the genomes of large DNA viruses. Here we demonstrate that direct in vitro ligation can be used to efficiently insert DNA fragments of up to 26,000 bp into the genome of vaccinia virus modified to contain a single NotI site either in the Escherichia coli lacZ gene or in the vaccinia virus thymidine kinase gene. Viruses containing chimeric genomes can be identified by chromogenic screening or thymidine-kinase-negative selection.