CONSTRUCTION AND CHARACTERIZATION OF VACCINIA DIRECT LIGATION VECTORS

Poxvirus vectors are extensively used as expression vehicles for prote in and antigen expression in eukaryotic cells. Customarily, the foreig n DNA is introduced into the poxvirus genome by homologous recombinati on. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily am enable for homologous recombination. We describe the construction and characterization of a new set of direct ligation vectors designed to b e universally applicable for the generation of chimeric vaccinia genom es. These vectors contain the pair of unique restriction sites Notl an d Apal to eliminate religation of poxvirus arms and fix the orientatio n of the insert DNA behind strongly expressing constitutive vaccinia p romoters. The insertion cassette has been placed at the beginning of t he thymidine kinase gene in vaccinia to use drug selection in the isol ation of recombinants. These viruses provide a set of universally appl icable direct ligation poxvirus cloning vectors, extending the utility of poxvirus vectors for construction and expression of complex librar ies. (C) 1997 Academic Press.