Construction of Recombinant Vaccinia Virus: Cloning into the Thymidine Kinase Locus

Vaccinia virus (VV) has proven to be a very useful tool for the expression and analysis of foreign gene products. The most common method used to produce recombinant viruses involves the insertion of foreign genes into the thymidine kinase (TK) gene of the VV via homologous recombination. This is accomplished through the construction of a recombination plasmid containing the VV TK gene into the middle of which the gene of interest is inserted, appended to an efficient VV promoter element of the desired temporal class. Confluent monolayers of cells are infected with wild-type VV and transfected with the plasmid DNA to allow homologous recombination to occur. This inactivates the endogenous TK gene-producing TK-negative virus that can be biochemically selected, and recombinants can be identified by a variety of screening methods.