Figure 4

Analysis of BAC clones after site-specific recombination. (a) HindIII restriction analysis of chloramphenicol/kanamycin double-resistant clones (pM24-BAC-CMV) after Cre-mediated integration between pM24-BAC and pFLS-CMV shuttle vector. Clones #3, 6, and 7 (marked with asterisks) show the expected restriction pattern. Clones #1, 2, 4, 5, and 8 contain an additional 7.8 kb fragment (closed arrowhead) and a greater amount of the 6.2 kb fragment (open arrowhead) which is consistent with the expected digestion pattern of pM24-BAC-CMV with doubly inserted shuttle vector [see Additional file 3]. Clone #10 also contains a greater amount of the 7.8 kb fragment, suggesting insertion of three or more copies of the shuttle vector. The remaining clone (#9) did not undergo recombination and shows a pattern identical to that of pM24-BAC. (b) HindIII restriction analysis of pM24-BAC-null clones obtained after Cre-mediated integration between pM24-BAC and pFLS-XICP4 shuttle vector. Clones #1, 2, 5, 9, 10 and 11 (marked with asterisks) show a restriction pattern consistent with singly integrated pM24-BAC-null. The other seven clones contain a partially deleted, incomplete HSV-BAC genome. The 8.2 kb HindIII fragment containing the BAC backbone (arrow) and the two neighboring fragments (20.1 and 2.2 kb; closed arrowheads) are preserved in all clones, and the 8.6 kb fragment (open arrowhead) adjacent to the 2.2 kb fragment is preserved in all clones except clone #13. Other fragments are lost in the deletion clones and new fragments of varying lengths are observed, suggesting that these deletions occurred randomly at different locations. For the HindIII restriction map, see Fig 2a and Additional file 3.