**1. Introduction**

102 Bacterial Artificial Chromosomes

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Bacterial artificial chromosomes (BACs) constitute minimal components of various whole genome-sequencing projects, including our own. The recent innovation of efficient recombinogenic bacterial strains allows systematic BAC modifications (i.e. recombineering; Reviewed in Copeland et al., 2001), setting BACs as an ideal experimental basis for functional genomics with their broad coverage of transcriptional regulatory elements. For instance, in the field of neuroscience, the GENSAT project extensively modified mouse BAC clones, covering gene transcriptional units preferentially expressed in the nervous system, and generated hundreds of BAC transgenic (Tg) mouse lines from those modified BACs (Gong et al., 2003). These BAC-Tg lines successfully recapitulated complex gene expression profiles in the nervous system (Gong et al., 2003), providing a rigid analytical platform so as to be able to answer the fundamental question of how tens of millions of neurons and thousands of cell-types can become elaborate interconnected circuitries in the brain by using only twenty-thousand sets of gene transcriptional activities.

Classic cadherins are adhesion molecules at the cell-cell adherence junction and the neuronneuron synapse peri-active zone (i.e. *puncta adherentia*) whose expression differentially delineates elaborated cytoarchitectures, such as layers and nuclei that constitute the basis for neural circuit formation in the vertebrate nervous system (Takeichi, 1995; Takeichi and Abe, 2005). Classic cadherins have 20 subclass members encoded by different genes, with each subclass harbouring distinct cell adhesiveness (Takeichi, 1995). In the *in vitro* aggregation assay system, it has been demonstrated that dissociated cells expressing the same sets of the classic cadherin subclass at a same level tend to make aggregates depending upon the calcium ion (Nose et al., 1988; Steinberg et al., 1994). Noticeably, in the developing nervous system, each classic cadherin subclass shows unique expression patterns, and such expression profiles are dynamically regulated during morphogenetic processes (Gumbiner, 2005; Redies, 2000). For instance, during chickens' early neural development, prospective neural tissue begins to express N-cadherin and, at the interface between the N-cadherin expressing cells and the surface ectodermal cells with E-cadherin expression, a Cadherin-6B positive, N-cadherin negative domain appears to segregate neural crest cells. Once they have emigrated from the neural plate/tube, neural crest cells finally begin to express Cadherin-7. This dynamical cadherin class switch is critical to the regulation of neurulation dynamics, since ectopically expressed N-cadherin perturbs the neural tube segregations from the ectoderm and/or the neural crest cell emigrations (Fujimori et al., 1990; Nakagawa & Takeichi, 1998). It is thus suggested that spatio-temporally regulated cadherin expression plays a pivotal role in animal morphogenetic processes.

While the physiological significance of differential cadherin expression profiles and/or cadherin class switches in neural development has been implicated, their gene regulatory mechanisms are largely unknown, due to the huge size and complex organisation of cadherin gene structures. Understanding the regulatory mechanisms for classic cadherin expression is crucial from a clinical point of view, as many cancer cells lose precise expression profiles of cadherins, resulting in the hyper-growth of cells and/or cell metastasis (Takeichi, 1995).

In our early studies, we have applied BAC-based technologies so as to screen gene regulatory patterns for a subclass of classic cadherins, cadherin-6 (*Cdh6*) whose gene structure is too large and complex for the identification of its promoter/enhancers by conventional methods. We succeeded in finding out that different genomic territories, located as far as 100-kbp upstream or downstream from the transcription start-site, is required for *Cdh6* expression at the defined time and place (Inoue et al., 2008a; Inoue et al., 2008b). Here, we extend the enhancer screening and reveal that a 6-kbp sized 3 prime intergenic region is critical in order to yield *Cdh6* expression along the somatosensory barrel, a distinct cytoarchitecture of the mouse cerebral cortex, at around postnatal day 7 (P7). Additionally, by taking advantage of the *Cdh6* enhancer/promoter activity identified, we establish a BAC Tg mouse line in which somatosensory barrels are stably illuminated by exogenous green fluorescent protein (GFP) expression, allowing us to suggest the roles of the retinoic acid (RA) related signalling pathway during cortical barrel field development and/or patterning. These results clearly demonstrate the strictly divisible *Cdh6* regulatory pattern along functional brain units, and the value of BAC-based experimental strategies in the field of developmental neuroscience.
