Fig. 1

Generation of BoHV-4-ΔIE1. a Schematic representation (not on scale) of the overall strategy to knock-down IE1 gene in BoHV-4 genome clone in SW 102 E. coli. Bo4 (orange), Bo5 [(IE1), blue] and Bo6 (green) gene coding regions are indicated by numbers, which represent nucleotide positions within the BoHV-4 genome [based on the BoHV-4-66p347 complete genome published sequence (GenBank accession number AF318573)]. The targeting fragment (red), IE1L-KanaGalK-IE1R, flanked by two homologous sequences allowed the insertion of the double selectable marker KanaGalK between the positions 19,672 and 20,229, deleting most of the IE1 coding region but leaving intact the Bo4 and Bo6 promoters. IE1 locus possess two PstI sites, one at the position 16,653 and one at the position 24,453, generating a fragment of 7800 bp. After targeting, a new PstI site, delivered by the targeted vector, was introduced into the IE1 locus and thus generating three PstI restriction sites and two PstI digestion product, of 4224 and 5251 bp respectively. PstI restriction enzyme digestion allowed to distinguish between targeted and untargeted clones (b), in fact the targeted clones (pBAC-BoHV-4ΔIE1) display two new bands (indicated by red arrows) respect to the untargeted control clone (pBAC-BoHV-4; indicated by the yellow arrow). This is further confirmed by southern blotting with a specific probe for KanaGalK (red circle). c Clonal stability of pBAC-BoHV-4ΔIE1 in SW 102 E. coli at passage 1, 5, 10, 15, 20 and analyzed by PstI restriction digestion