In vivo gene transfer studies on the regulation and function of the vasopressin and oxytocin genes

Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one-cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis -acting sequences mediating the regulation of the vasopressin-oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell-specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High-throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication-deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long-lasting expression of biologically active proteins in vasopressin and oxytocin MCNs.