Molecular Approaches to Study Neuronal Cell Types and their Connections in Mice

Genomic approaches can be used to visualize specific neuronal cell types including their axonal projections. Most of the mouse genome is cloned as large fragments (150-200 kilobases in size) in so-called bacterial artificial chromosomes (BACs) — vectors that can be easily propagated inside cells. The advantage of BACs is that they are usually large enough to carry both the coding region of a gene and its regulatory sequences, which determine where and when the gene is expressed. Using a library of BACs, one can systematically replace the coding regions of different genes with a 'reporter' sequence that encodes a fluorescent protein (enhanced green fluorescent protein, or EGFP). By injecting a modified BAC into a mouse egg, one can generate transgenic animals in which we can study the spatiotemporal expression patterns of each gene in the central nervous system. This approach is currently being pursued in the Gene Expression Nervous System Atlas (GENSAT) BAC Transgenic Project (www.gensat.org), which has already provided data on some 150 genes, but aims to provide detailed expression maps for many more (Gong et al. 2003). The BAC method still faces several problems such as specificity when used with very large genes whose regulatory sequences do not fit into a single BAC, or sensitivity where several copies of a BAC are required to make the fluorescent signal strong enough to detect it.

Where EGFP is expressed throughout the entire neurons including the axon, this approach has the potential to trace projections of neurons that are characterized by their molecular genetic properties rather than by their morphological and physiological appearance. For example, the GENSAT project has revealed cell classes specific to individual brain regions and to the different layers of the cerebral cortex. Visualizing their axonal projections in 3D space will be required to map the connectivity patterns of genetically characterized neurons.

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