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Bacterial Artificial Chromosomes (BACs) had been minimal components of various genome-sequencing projects, constituting perfect analytical basis for functional genomics. Here we describe an enhancer screening strategy in which BAC clones that cover any genomic segments of interest are modified to harbor a reporter cassette by transposon tagging, then processed to carry selected combinations of gene regulatory modules by homologous recombination mediated systematic deletions. Such engineered BAC-reporter constructs in bacterial cells are ready for efficient transgenesis in mice to evaluate activities of gene regulatory modules intact or absent in the constructs. By utilizing the strategy, we could speedily identify a critical genomic fragment for spatio-temporally regulated expression of a mouse cadherin gene whose structure is extraordinarily huge and intricate. This BAC-based methodology would hence provide a novel screening platform for gene transcriptional machineries that dynamically fluctuate during development, pathogenesis and/or evolution.

The mammalian cerebral cortex can be tangentially subdivided into tens of functional areas with distinct cyto-architectures and neural circuitries; however, it remains elusive how these areal borders are genetically elaborated during development. Here we establish original bacterial artificial chromosome transgenic mouse lines that specifically recapitulate cadherin-6 (Cdh6) mRNA expression profiles in the layer IV of the somatosensory cortex and by detailing their cortical development, we show that a sharp Cdh6 gene expression boundary is formed at a mediolateral coordinate along the cortical layer IV as early as the postnatal day 5 (P5). By further applying mouse genetics that allows rigid cell fate tracing with CreERT2 expression, it is demonstrated that the Cdh6 gene expression boundary set at around P4 eventually demarcates the areal border between the somatosensory barrel and limb field at P20. In the P6 cortical cell pellet culture system, neurons with Cdh6 expression preferentially form aggregates in a manner dependent on Ca(2+) and electroporation-based Cdh6 overexpression limited to the postnatal stages perturbs area-specific cell organization in the barrel field. These results suggest that Cdh6 expression in the nascent cortical plate may serve solidification of the protomap for cortical functional areas.

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