Figure 4

BoHV-4 gB disruption. A) Overall strategy to delete a 1261 bp sequence from the ORF8 coding for gB, via heat inducible homologous recombination. The 2232 bp Kana-GalK selectable DNA stuffer, flanked by ORF8 homologous regions, was introduced between positions 11640 and 12901 of the BoHV-4-A strain cloned as a BAC. The expected ORF8 locus (A, bottom) has an increased size of the HindIII fragment (5280 instead of 4314 bp), generated by HindIII restriction enzyme digestion. The diagrams here presented are not to scale. B) HindIII Restriction profile and corresponding Southern Blotting of six representative targeted clones, compared to the untargeted control. Southern Blotting was performed with a probe spanning Kana sequence and confirmed the above data. C) Clonal stability of the pBAC-BoHV-4-A-ΔgBKanaGalK in Escherichia coli SW102 cells, passaged for 25 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis.