**3.3 BACs in the field of neuroscience research**

In the present study, BAC-based methodology enabled us to systematically evaluate intricate genetic machineries in the mouse brain, highlighting the value of BAC usage in the field of neuroscience research. Recently, others have also developed many useful BACbased strategies, and here we discuss the advantages and/or future potential of some of these strategies in the field of neuroscience research.

First, we have realised that BACs must provide a useful and ideal basis in approaching the complex gene regulatory machinery that elaborates the nervous system. For instance, a difficulty involved in studying the vertebrate nervous system has lain in its complexity and, when we try to rigorously dissect its specialised structure and function, it should be an essential step in discriminating a defined group of cells among numerous neurons and glial cells. As has been demonstrated by the present study, homologous recombination-based

mouse founders (Inoue et al., 2009). In the Tg cerebral cortex, we found that GFP expression shows exactly the same patterns as with the original LacZ-Tg mice (Fig 1 and 2). In particular, this GFP-BAC-Tg mouse line illuminated the S1 barrel structure of the whole mount brain preparations, allowing us to easily image the S1-barrel territory whose identification generally requires specific histological staining processes, such as the CO

We then administrated RA to the mothers or pups for GFP-BAC-Tg, with the concentration reported to induce abnormality in the cortex (Smith et al., 2001) and evaluated how RA affects the GFP expression patterns at P7. As a result of this, no drastic change was observed for the cortical barrel patterning when RA was administrated later than embryonic day 17.5 (E17.5). However, RA administration at E14.5 resulted in massive perturbation of S1 barrel patterning, with ambiguous area boundaries illuminated by GFP expression. Noticeably, the intensity of the GFP expression appeared to be decreased due to the qualitative and quantitative differences among *Cdh6*::*GFP* positive cortical cells and/or thalamocortical axon terminals. These results imply that the role of RA in regulating *Cdh6* expression and/or cortical area pattering is just limited to

It is now widely accepted that cortical area patterning begins as early as mouse E12.5 when the counter-gradient of the transcription factors Pax6 and Emx2 is established in the cortical ventricular zones (Bishop et al., 2000; Hamasaki et al., 2004). This gradation pattern, as generated by such secreted molecules as *Fgf8*, is shown to be the basis of cortical arealisation yet other transcriptional factors, such as *Coup-TFI,* could regulate the area-specific differentiation of distinct subtypes of cortical neurons independently of *Fgf8*-*Pax6*/*Emx*2 gene functions (Armentano et al., 2007; Fukuchi-Shimogori & Grove, 2001, 2003). Our results, together with a previous series of studies, thus suggest that RA accumulated earlier than E14.5 might play a role in cortical arealisation by affecting the production, migration, positioning and/or circuit formation of the cortical S1 barrel layer IV neurons that eventually express *Cdh6*. The next critical step would be to examine whether RA-related signalling could be interactive with the 5,884 bp territory identified in this study that

In the present study, BAC-based methodology enabled us to systematically evaluate intricate genetic machineries in the mouse brain, highlighting the value of BAC usage in the field of neuroscience research. Recently, others have also developed many useful BACbased strategies, and here we discuss the advantages and/or future potential of some of

First, we have realised that BACs must provide a useful and ideal basis in approaching the complex gene regulatory machinery that elaborates the nervous system. For instance, a difficulty involved in studying the vertebrate nervous system has lain in its complexity and, when we try to rigorously dissect its specialised structure and function, it should be an essential step in discriminating a defined group of cells among numerous neurons and glial cells. As has been demonstrated by the present study, homologous recombination-based

staining method.

those embryonic stages earlier than E14.5.

contains *RORbeta* related transcription factor binding motifs.

**3.3 BACs in the field of neuroscience research** 

these strategies in the field of neuroscience research.

systematic deletions from BAC clones combined with efficient mouse trans-genesis now allows for the quick identification of *cis*-regulatory elements from the huge *Cdh6* gene locus, which contrasts with the conventional methodology where hundreds of conserved genomic regions must be evaluated one-by-one by means of plasmid-based methods (See discussions in Asami et al., 2011). A problem with this recombination-based method might lie in its limit for BAC clone usage, since the BAC clone for recombination must always include an ATG translation initiation codon for the gene of interest to achieve in-frame integration of the reporter cassette. This being the case, *cis*-regulatory elements located far outside of the ATGcontaining BAC clone cannot also be treated by this method. However, if combined with the transposon-based BAC modification method, one can reliably monitor the gene transcriptional activity of any BAC clones, regardless of their gene/ATG-exon coverage (Asami et al., 2011). Hence, a BAC-based strategy would greatly help the understanding of the upstream/downstream relationships among thousands of genes that are preferentially expressed in the nervous system, revealing the entire genetic programmes for building up the nervous system. Noticeably, many human single nucleotide polymorphisms (SNPs) that are tightly linked to genetic disorders have been identified in the intergenic regions (Wang et al., 2009). These SNPs are thought to play roles in regulating gene expression and/or chromosomal structures, yet the methodologies that can reliably detect their functional significance are limited in number at the present time. In this context, our BAC-based methodology could immediately serve as a steadfast platform in approaching such critical research subjects (Asami et al., 2011).

Secondly, we have demonstrated that BAC-based methodology is very useful for the stable and efficient labelling of distinct cells-types during neural development. Since fluorescent proteins – such as GFP – can easily visualise cell morphology, they have been broadly used, from basic molecular-cell biology to biomedical studies. In the field of neuroscience research, it remains a fundamental issue to thoroughly identify the original location of neurons and their partners among the billions of neurons, since they often make contact with other neurons located far away from the soma. For this purpose, the use of a membrane localisation signal, such as GAP43 tagged GFP, would clearly visualise axons, while nuclear localisation signal-based reporter introduction would precisely identify the soma location. Recently, a rabies virus mediated single synaptic transfer event was applied so as to enable systematic labelling of both the starter neurons and their primary partners in connections (Miyamichi et al., 2011). However, in order to restrict the number of neurons labelled by this method in the nervous system, it is most critical to identify enhancers/promoters that confine gene expression in the limited population of cells amongst the tens of thousands of neurons. In this context, BACs do provide an ideal resource for such an analysis because we can now easily engineer a given BAC clone to include sets of enhancers/promoters for obtaining restricted gene expression profiles, and the GENSAT project indeed generated hundreds of BAC transgenic mouse lines that differentially illuminate specific sets of cells in the nervous system (Gong et al., 2003; Gong et al., 2010). If a GFP tagged L10a ribosomal protein is expressed by BAC trans-genesis, the mRNA expressed in the specific set of cells should be illuminated and can further be isolated by the fluorescent activated cell-sorting system so as to profile their molecular characteristics (Heiman et al., 2008).

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 complex cellular organisation.

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 research in the field of neuroscience.
