A vaccinia virus transfer vector using a GUS reporter gene inserted into the I4L locus

A vaccinia virus (VV) transfer vector is described which enables integration of heterologous sequences into the I4L locus (ribonucleotide reductase-encoding gene) through co-insertion of a GUS selection marker. I4L- VV recombinants formed blue plaques when an agarose overlay containing XGluc (5-bromo-4-chloro-3-indolyl-beta-glucuronide) was added to the infected cell monolayer. Viruses already containing a lacZ reporter gene were also suitable recipients for the selection procedure since infection with a VV lacZ recombinant did not produce any blue plaques with XGluc. The addition of a synthetic early promoter downstream from the GUS cassette initiated the predicted-size transcript during an infection. Insertion of genes with VV p7.5-promoters into the I4L, J2R and K1L loci of the same virus produced viable virus recombinants even though recombination between these loci could be demonstrated. These techniques should be valuable for the further development of VV as a polyvalent vector.