**5. Choice of the two hosts for BAC engineering**

The use of BACs addresses different aims and goals in different cellular systems and some disadvantages must still be overcome. One seeming disadvantages is that our *B. subtilis* system is still too new for wide dissemination. Examples of BACs that have been manipulated in BGM systems to date are listed in Table 1. Advantages are that the *B. subtilis* host system is obviously superior to BAC technologies that make use of the *E. coli* system (Fig. 2). We focused on using BACs in our mouse genetics studies; investigations of gene loci other than *jmj* (Fig. 6b) are in progress. Another advantage is that a total of 9 antibiotic resistance gene cassettes are currently available for *B. subtilis* selection: neomycin (*neo*), spectinomycin (*spc*), chloramphenicol (*cat*), tetracycline (*tet*), erythromycin (*erm*), blasticidin S (*bsr*), kanamycin (*kam*), phleomycin (*phl*), and hygromycin (*hyg*). These antibiotic resistance genes will greatly facilitate the development of multipurpose BGM vectors that allow all desired manipulations. Although examples for the practical application of BAC technologies remain limited, we are in the process of producing a BAC-BGM kit that involves a simple protocol. The next generation of BGM should be accompanied by handy manuals so that even non-expert users of *B. subtilis* can perform routine experiments based on a basic understanding of the principles that are the foundation of BGM. The rapid preparation and assembly of fragments in the host is key for simplifying necessary procedures and for shortening the time required for the production of engineered fragments. From this perspective, we think that the BGM system is an invaluable tool for the creation of accurately designed guest DNA that does not rely on existing BAC modification kits in *E. coli*. However, the role of *E. coli* as an initial producer of the BACs prior to BGM will remain unaltered. Initially, our new BAC vectors, p108BGMC(*cat*) and p108BGME(*erm*), can be expected to replace the currently widely-used BAC vectors that lack markers for BGM.


\*1BAC clones in literature \*2Estimated by gel electrophoresis. \*3Confirmed by Southern blot analysis using original BAC clone as probes. \*4Used for transformation of *E. coli* strains DH1 or DH10B. Refereed by †1 Kaneko et al., 2005, †2 Kaneko et al., 2003 †3 Kaneko et al., 2008, and †4 Itaya et al., 2000.

Table 1. BAC clones\*1 handled by BGM systems
