Disorders of CNS function place an incalculable emotional and financial burden on our resources as a society. Further investigation of nervous system development and of novel neural genes may lead directly to the treatment of CNS disorders. Recently my colleagues and I have begun to develop methods for isolating new nervous system genes via the neural differentiation of embryonic stem (ES) cells in vitro. This system, which leads to functional neurons and glia in culture, also recapitulates embryonic nervous system development. In Pilot studies, we have determined that ES cell neural differentiation can be used to isolate novel genes that are temporally and spatially expressed in the embryonic CNS. Furthermore, as ES cells are easily genetically manipulated, we have begun to utilize GFP (green fluorescent protein) gene targeting to generate ES cell lines that are marked in specific neural lineages. Using this approach, specific neurons or glia can be isolated via flow cytometry. We plan to develop new technologies for the isolation and expressioncharacterization of novel genes within the CNS, using the following specific aims: 1) Develop technology for the isolation and characterization of novel neuroembryonic genes; 2) Enhance gene targeting methods for generating neuralspecific ES cell """"""""knockin"""""""" panels; and 3) Develop technology for generating celltype specific cDNA libraries of the CNS. By combining the powerful approaches of ES cell neural differentiation and gene targeting, we will be able to identify and characterize the expression of genes that influence CNS development. These technologies may lead to the discovery of genes involved in human disease, and of pharmaceuticals which influence neural degeneration and regeneration. We anticipate the real possibility of enriching and purifying specific neurons and glia for cytotherapeutic applications. Should the recently isolated human ES cells differentiate in a similar manner, the therapeutic value of these technologies may be even greater.
