Figure 4

Characterization of mutants of OV-IA82Δ113 and OV-IA82Δ116 by Southern blotting. A. OV-IA82Δ113. Upper panel is a schematic of OV-IA82 genome before and after removal of the 113 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ113. Lower panel shows Southern blot analysis. Genomic DNA was isolated from OV-IA82 (lane 1) and OV-IA82Δ113 (lane 2) and digested with restriction enzyme AflII or EcoRI respectively. The 113 internal probe was unable to detect the 113 gene in the recombinant OV-IA82Δ113 genome, indicating that the 113 gene was completely removed from the 113 locus of the genome. The 001 probe detected both end of the 001 loci in both OV-IA82 and OV-IA82Δ113 genomes. B. OV-IA82Δ116. Upper panel A is a schematic of OV-IA82 genome before and after removal of the 116 gene using the deletion vector, pSPV-EGFP by double homologous recombination to generate gene-deletion mutant OV-IA82Δ116. Lower panel shows the 116 gene, which was completely removed from the 116 locus of the OV-IA82 genome by Southern blot analysis. The 116 internal probe was unable to detect the 116 gene in the OV-IA82Δ116 genome. The 001 probe detected both end of sequences in OV-IA82 (lane: 1) and three different clones of OV-IA82Δ116 (lanes: 2 to 4).