Cloning and various modification methods of the BGM vector system in the generation of transgenic mice. (A) The cloning strategy used to introduce BAC inserts into the BGM vector (Figure 2). BGM vectors possess two antibiotic gene cassettes: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The cI-spc is flanked by two BAC vector sequences that function as the cloning site for BAC inserts. The original BGM strains are resistant to Spc and sensitive to Nm. Once a BAC insert is cloned into the BGM vector, the recombinant becomes resistant to Nm and sensitive to Spc because the cI-spc cassette is replaced by the BAC insert. (B) Modifications of large DNA fragments. The BGM vector system allows various manipulations, including insertion (e.g., EGFP is designated as “G” in the box) (Figure 3), deletion and the inversion (Figure 4) and fusion of two overlapping clones (Figure 5). (C) The BGM inserts can be retrieved by several methods, and the recombinant DNA fragments can be used for the generation of transgenic animals by microinjection (Figure 6). The closed triangles represent a target gene and its direction. The BAC vector sequences are indicated by the closed and open arrows. The half part of BAC vector containing chloramphenicol resistance gene for E. coli is depicted by open arrow (defined as right side). The I-PpoI sites, designated as “I” are introduced to flank the BAC vector sequences. The deletion method is described in .