Abstract
The supraoptic nucleus (SON) is a particularly good model for the study of cell-type specific gene expression because it contains two distinct neuronal phenotypes, the oxytocin (OT) and vasopressin (AVP) synthesising magnocellular neurones (MCNs). The MCNs are found in approximately equal numbers and selectively express either the OT or the AVP gene in approximately 97% of the MCN population in the SON. An unresolved issue has been to determine what mechanisms are responsible for the highly selective regulation of the cell-type specific expression of OT and AVP genes in the MCNs. Previous attempts to address this question have used various bioinformatic and molecular approaches, which included using heterologous cell lines to study the putative cis-elements in the OT and AVP genes, and the use of OT and/or AVP transgenes in transgenic rodents. The data from all of the above studies identified a region < 0.6 kbp upstream of OT exon I and approximately 3 kb upstream of AVP exon I as being sufficient to produce cell-specific expression of the OT and AVP genes, respectively, although they failed to identify the specific cis-domains responsible for the MCN-specific gene expression. An alternative experimental approach to perform promoter deletion analysis in vivo (i.e. to use stereotaxic viral vector gene transfer into the SON to further dissect the cis-elements in the OT and AVP genes) will be described here. This in vivo method uses adeno-associated viral (AAV) vectors expressing OT-promoter deletion constructs and utilises the enhanced green fluorescent protein (EGFP) as the reporter. The AAV constructs are stereotaxically injected into the rat brain above the SON and, 2 weeks post injection, the rats are sacrificed and assayed for EGFP expression. Using this method, it has been possible to identify specific regions upstream of the transcription start site in the OT and AVP gene promoters that are responsible for conferring the cell-type specificity of the OT and AVP gene expression in the SON.