Neuropeptide gene transfer into neuronal and glial cell lines

Procorticotrophin-releasing hormone (ProCRH) is the precursor to the hypothalamic neuropeptide CRH(1-41) which mediates the neuroendocrine response to stress. In neuroendocrine cells and neurones, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of the cellular processes where they are stored until they are released upon an external stimulus. In order to study the post-translational processing and intracellular trafficking of neuropeptides in neuronal cells we have established stably transfected Neuro2A cells and NG115-401L cells expressing proCRH. The expression vector used contains glutamine synthetase (GS) coding sequences which are used as a dominant selectable marker in cells already containing GS genes. The various clonal cell lines isolated express different levels of CRH as assessed using a specific radioimmunoassay (RIA) thus indicating that the level of expression of the exogenous gene must depend upon the site of chromosome integration. Using immunofluorescence labelling, we have demonstrated that in Neuro2A and NG115-401L cells proCRH is packaged in vesicles which accumulate at the tips of cellular processes.