**2.1 BAC modification via homologous recombination in bacterial cells**

The recombinogenic bacterial strain *EL250* (Lee et al., 2001) was used in the present study. For the homologous recombination, ~1-kbp homology arms were amplified from the BAC clone of interest (=*RP23-78N21* in this study) by means of a polymerase chain reaction (PCR), and were cloned into a conventional TA cloning vector (pGEM-T-Easy; Promega). For homologous recombination, beta-galactosidase (*LacZ*) and a growth-associated protein-43 tagged enhanced green fluorescent protein (GAP43-EGFP) gene expression cassette were inserted into the BAC clone of interest; a *LacZ*/GAP-EGFP gene expression cassette and a gene cassette for clone selections (e.g. ampicillin resistant gene, kanamycin resistant gene, etc.) were cloned in between the homology arms and the fragment containing arms as well as reporter/selection gene cassettes which were purified by means of agarose gel electrophoresis after complete digestion with proper restriction enzymes. For homologous recombination mediated deletion of given territories from the BAC clone of interest, only the selection cassette was cloned in between the homology arms and the portion containing

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

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.

**2.1 BAC modification via homologous recombination in bacterial cells** 

The recombinogenic bacterial strain *EL250* (Lee et al., 2001) was used in the present study. For the homologous recombination, ~1-kbp homology arms were amplified from the BAC clone of interest (=*RP23-78N21* in this study) by means of a polymerase chain reaction (PCR), and were cloned into a conventional TA cloning vector (pGEM-T-Easy; Promega). For homologous recombination, beta-galactosidase (*LacZ*) and a growth-associated protein-43 tagged enhanced green fluorescent protein (GAP43-EGFP) gene expression cassette were inserted into the BAC clone of interest; a *LacZ*/GAP-EGFP gene expression cassette and a gene cassette for clone selections (e.g. ampicillin resistant gene, kanamycin resistant gene, etc.) were cloned in between the homology arms and the fragment containing arms as well as reporter/selection gene cassettes which were purified by means of agarose gel electrophoresis after complete digestion with proper restriction enzymes. For homologous recombination mediated deletion of given territories from the BAC clone of interest, only the selection cassette was cloned in between the homology arms and the portion containing

plays a pivotal role in animal morphogenetic processes.

**2. Protocols** 

arms, and the selection cassette was isolated as described above. The selection cassette was put in between two *FRT* sites for its eventual excision by the inducible *Flpe* gene (see below).

The recombination system in *EL250* cells (Lee et al., 2001) was activated via heat shock, and their electroporation-competency (EP) was conferred on ice so as to maintain the recombinogenic activity. Briefly, *EL250* cells that harbour the BAC clone of interest were precultured in 50 ml of LB without antibiotic at 32C, until an optical density of 600 nm (OD600) was reached at 0.5. By adding 2.5 ml of the overnight saturated culture to 50 ml of LB, it normally took ~90 min to obtain 0.5 OD600 in the shaking culture at 32C. Then, 15 ml of the culture was heat-shocked at 42C for 15 min in a water bath with the shaker's agitation, while another 15 ml was maintained in the shaking culture at 32C as the control. Both of the cultures were subsequently placed on ice for 15min, centrifuged, and quickly washed three times by ice-cold water in order to draw the electro-competency for the heat-shocked cells. A few hundred nano-grams of the resultant purified fragment was then electroporated with a 1 mm gap cuvette by 1.8 kV (BioRad) into the EP-competent cells, pre-incubated for 2 hours at 32C, and selected on an LB agar-plate containing 12.5 g/ml chloramphenicol and 12.5 g/ml Kanamycin/Ampicillin for ~40 hours at 32C. Starting with the culture of *EL250* cells which harboured the BAC clone of interest, from 15 ml we obtained ~100 colonies from the heatshocked cell plate after the EP, with more than 90% of the colonies containing precisely the modified BAC clone. To excise the selection cassette from the modified BACs, arabinose was added to the log phase liquid culture of the selected clone, with the correct recombinant BACs at 0.1% v/v for 1 hour at 32C so as to sufficiently induce the *Flpe* gene in *EL250* cells. The loss of the selection cassette was monitored on the L-plate containing the antibiotic for the selection cassette. Homologous recombination events were always verified by PCR and/or electrophoresis after purification of the BAC DNA from each colony, as mentioned below.
