Overall strategy of BoHV-4CMV-IgKE2-14ΔTK generation. a) First targeting, where kanamycin resistant cassette (Kana) adjacent to the galactokinase cassette (GalK) and flanked by BoHV-4 thymidine Kinase gene and adjacent sequences (TK), are introduced into the TK gene of BoHV-4 genome cloned as bacterial artificial chromosome (BAC-BoHV-4) via heat inducible homologous recombination in SW 102 E. coli. Following a positive selection on (solid) minimal plates containing galactose as the only source of carbon and a positive selection on (liquid) medium containing Kanamycin. Selected clones, fermenting lactose (red colonies) on McConkey plates (representative plate). b) HindIII restriction enzyme analysis of 6 clones out of hundreds, all displaying the right targeting, where a new 2.6 kb band (indicated by arrow, 1 to 6) corresponding to the insertion of the Kana/GalK cassettes is present but missing in the untargeted control. c) Second targeting and counter selection, where the Kana/GalK cassettes were removed via heat inducible homologous recombination and replaced with CMV-IgKE2-14 expression cassette. All colonies tested through HindIII restriction enzyme analysis and agar gel electrophoresis (d) shows the right re-targeting. The disappearing of the 2.6 kb band and the appearing of a new band with a lower molecular size are indicated by arrows.