**4. Conclusions**

114 Bacterial Artificial Chromosomes

Thirdly, a BAC-based methodology is highly expected to open a new window into the study of unknown processes for brain development and/or the functional dynamics of neural circuitries. For instance, in multicellular model organisms, such as the mouse, the fly and the nematode, loss of function studies are currently the gold standard for revealing given gene functions, contributing to the revelation of genetic programmes at the early developmental stages. There had been, however, a problem in that a simple loss of function analysis sometimes results in early embryonic lethality, preventing researchers from evaluating the gene functions in mature organs, such as brains. To circumvent this situation, a conditional gene knock-out strategy was established so that a given gene function is abolished at a defined time and place by genetically introducing the enhancer-driven site-specific recombinase *Cre* and its recognition sites LoxP sequences into the gene locus of interest. Considering their extensive coverage of various enhancers/promoters in the genome of multicellular model organisms, BAC clones should serve as the perfect starting points in the establishment of useful driver transgenic animals for conditional knock-out studies. Additionally, if the expression cassette for *Cre* and the estrogen receptor T2 variant fusion protein (*CreERT2*; Feil et al., 1997) is integrated into a proper BAC clone to generate Tg animals in which *CreERT2* proteins are expressed among a limited group of cells, one can precisely control the timing to generate gene mutant cells by merely administrating tamoxifen, which allows selective *CreERT2* localisation into the cell nuclei so as to excise the gene of interest by recombination. *CreERT2*-Tg animals might further be suitable for genetic cell-lineage tracing. In the mouse system, this can generally be achieved by using the reporter mouse lines, such as *Rosa26R*, in which *LacZ* reporter expression is suppressed by the intercalation of a stopper put in between the LoxP sequences. When this reporter line is mated with the *CreERT2*-Tg mouse line, the stopper is excised only with the administration of tamoxifen and, thereafter, a limited population of cells will be genetically and permanently marked by the reporter expression. Indeed, we have generated the *Cdh6*::*CreERT2*-BAC-Tg mouse to clarify the relationship between the *Cdh6* gene expression boundary at the early cortical plate and the mature areal boundary, and have found a rigid correlation (Terakawa et al., manuscript submitted). Useful *CreERT2*-Tg mouse lines could be further be mated with the recently created Brainbow mouse Tg line, logically allowing the genetic labelling of individual cells in the nervous system by different fluorescent colour combinations (Livet et al., 2007). Such spatio-temporally regulated labelling of cells must aid in unveiling the functional dynamics of the nervous system in higher vertebrates with

To finally address the fundamental question as to how the elaborated neural circuitries work in the *in vivo* context, the BAC-based introduction of optogenetic probes, such as channel rhodopsin and halorhodopsin, into specific sets of neurons might make it possible to selectively switch on and off neuronal activities within the regions that receive the relevant light stimuli (O'Connor et al., 2009; Zhang et al., 2007). Since the individual optogenetic probe harbours different wavelengths' selectivity, defined sets of neuronal and/or muscle activities can be manipulated by simply applying combinatorial light stimuli to the Tg animals. This technology therefore speeds the detailing of which circuitries are actually responsible for a given behaviour and/or the process for learning and memory, exemplifying how BAC-related experimental methods can be applicable to wide range of

complex cellular organisation.

research in the field of neuroscience.

Taking advantage of systematic BAC modification methodologies via homologous recombination and/or transposon tagging in bacterial cells, as well as efficient BAC transgenic strategies in various multicellular organisms, such as mice, it is now possible to mark and manipulate a given gene function amongst restricted cell groups in the nervous system at will. Given that BACs constitute the minimal components of various whole genome-sequencing projects, BAC-based technology would significantly facilitate the detail of entire genetic programmes that elaborate the complex structure and function of the nervous system, including our own. Such detailed information would greatly help the appreciation of the intricate principles of neural evolution and development, encoding, processing and/or pathogenesis.
