Additional file 5.
Figure S5. A) Overall strategy to reconstitute the ORF8 complete gB locus, via heat inducible homologous recombination. The 2911 bp gB locus, amplified by PCR (between positions 10576 and 13487 of BoHV-4-A genome), was introduced to replace the 2232 bp Kana-GalK selectable DNA stuffer, flanked by ORF8 homologous regions in the BoHV-4-A ΔgBKanaGalK strain cloned as a BAC. The expected ORF8 locus (A, bottom) has decreased size of the HindIII fragment (4314 instead of 5280 bp), generated by HindIII restriction enzyme digestion (diagram not on scale). B) HindIII restriction profile and corresponding Southern Blotting of five 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-ΔgBrevertant in Escherichia coli SW102 cells, passaged for 25 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis.
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Franceschi et al. BMC Veterinary Research 2013 9:6 doi:10.1186/1746-6148-9-6