**4.2 Genetic dissection of the guest portion from the** *B. subtilis* **genome**

The second method, genome dissection, largely depends on *B. subtilis* genetic systems. Although this method appears complex at first glance because of the molecular apparatus briefly described in Fig. 7, it is in fact simple. Intrachromosomal homologous recombination between the two DNA repeats makes it possible to physically disconnect the BAC segments. As the disconnected DNA was designed to carry a DNA replication origin site (*oriN*) different from *oriC* of the chromosome, it starts replicating autonomously as a plasmid independent from the chromosome. Itaya and Tanaka (1997) reported a 300-kbp DNA fragment that manifested superb genetic stability. Despite the potential to produce circular

than several hundred kbp has been difficult because of its fragility in test tubes. However, its use for microinjection into, for example, fertilized mouse eggs requires pure and undamaged DNA. We implemented and tested various steps to isolate undamaged giant DNA maintained in liquids. Our improved protocol that involves 2 gel electrophoresis runs and collection on a dialysis membrane made it possible to concentrate undamaged giant DNA. The successful purification of up to 220 kb mouse genomic DNA carrying reporter genes facilitated the creation of transgenic mice (manuscript in preparation). Because our

1. Fragmentation using endonucleases is simple. Improvements for the isolation step are

2. Only one report describing genome dissection has been published (Itaya & Tanaka,

3. For recombinational transfer see the text and refer to Tsuge & Itaya (2000) and Itaya & Tsuge (2011). Although the process appears to be complicated, the BAC insert can be copied in the BGM and pasted into a linear plasmid to complete circularization. This

The second method, genome dissection, largely depends on *B. subtilis* genetic systems. Although this method appears complex at first glance because of the molecular apparatus briefly described in Fig. 7, it is in fact simple. Intrachromosomal homologous recombination between the two DNA repeats makes it possible to physically disconnect the BAC segments. As the disconnected DNA was designed to carry a DNA replication origin site (*oriN*) different from *oriC* of the chromosome, it starts replicating autonomously as a plasmid independent from the chromosome. Itaya and Tanaka (1997) reported a 300-kbp DNA fragment that manifested superb genetic stability. Despite the potential to produce circular

1997). The potential for isolating DNA above 300-kbp is described.

**4.2 Genetic dissection of the guest portion from the** *B. subtilis* **genome** 

process yields a plasmid carrying the copied DNA segment.

method is technically simple it will be of great value in future studies.

Fig. 7. Three methods to retrieve BAC-DNA from BGM.

described in the text.

DNA larger than 300 kbp by this method, its application has been restricted by the rather complicated procedures involved in its creation (Itaya, M., unpublished observations).
