Figure 5

(A) 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-U genome cloned as bacterial artificial chromosome (BAC-BoHV-4-U) 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. b) HindIII restriction enzyme analysis of 2 clones out of hundreds, displaying the right targeting, where a new 2.6 kb band (indicated by arrow) corresponding to the insertion of the Kana/GalK cassettes is present but missing in the untargeted control. Southern hybridization with a Kanamycin specific probe confirms the specific targeting.