Cell‐Specific Expression of the Rat Oxytocin Gene in Transgenic Mice

Abstract

The genes for the posterior pituitary hormones oxytocin (OT) and vasopressin (VP) are expressed in magnocellular neurons of the hypothalamus. Previous attempts to obtain cell-specific expression of OT and VP transgenes in mice have been unsuccessful using constructs containing only the OT or VP genes. As the endogenous genes are located near each other on the same chromosome, we investigated four transgenic mouse lines incorporating a single 5.2 k bp construct of rat genomic deoxyribonucleic acid with 1.63 k bp and 3.55 k bp of the OT and VP genes, respectively. Rat OT transcripts were detected only in transgenic mouse OT neurons of the paraventricular and supraoptic nuclei in two lines. The levels of endogenous and transgene OT transcripts increased in response to 10 days of lactation. Furthermore, rat OT-associated neurophysin immunoreactivity was detected in magnocellular cell bodies as well as in the posterior pituitary of the transgenic but not the control mice. Cell-specific rat VP expression was not detected in these transgenic mice.The oxytocin (OT) and vasopressin (VP) genes code for related nonapeptides that participate in the regulation of parturition and lactation, and fluid balance, respectively (1). Transcripts encoding the preprohormones are made primarily in separate populations of magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. The preprohormones are processed into OT or VP and associated neurophysins (NP-OT, NP-VP) during axonal transport to the posterior pituitary where the products are secreted into the circulation (2). O T mRNA and serum peptide levels increase with parturition, lactation, and, along with VP mRNA and serum peptide levels, with hyperosmolality (reviewed in (I) and (3)).Little is known about factors that trans-activate the OT gene or cis-acting sequences (response elements) that allow control of expression. An estrogen responsive element has been detected in the human OT S'-flanking region between bases -164 and -1 15 (4). Similar elements in rat and human OT 5'-flanking regions have also been described by Burbach et al. (5). The latter authors detected no estrogen receptors or changes in OT mRNA levels there following ovariectomy and replacement with estradiol, although Miller et al. (6) described lower levels in ovariectomized or prepubescent female rats. In addition, OT neurons concentrate estradiol (7).Interactions between trans-acting factors and response elements found in vitro need to be validated in vivo. Transgenic animals have been used for this purpose (8). To date, however, few attempts have been made to study the expression of OT in transgenic mice. Carter and colleagues produced transgenic mice that contained 4.2 k bp of bovine genomic DNA, including the OT transcriptional unit and 0.6 k bp upstream (9). No bovine OT expression was detected in the mouse hypothalamus, but there was ectopic expression in the cerebellum, lung and testis. The cell-specific regulation of VP expression is also not understood. ...

Mapping of the Bovine Oxytocin Gene Control Region: Identification of Binding Sites for Luteal Nuclear Proteins in the 5′ Non‐Coding Region of the Gene

“…However, transfections of these cells with gene constructs encompassing up to 3.2 k b of the tentative bovine oxytocin promoter region have shown only a very low basal activity, but no transcriptional up-regulation of the transfected gene under culture conditions that lead to up-regulation of the chromosomal oxytocin gene (Walther, unpublished results). Possibly elements further away from the oxytocin coding region are necessary for up-regulation of transcription, as has been suggested from studies in transgenic mice (12,13). In the study presented here we have taken an alternative in vitro approach to analyse thc regulatory role of sequences from the 5' non-coding region of the bovine oxytocin gene.…”

Bovine Oxytocin Transgenes in Mice

“…Thus, sequences within the OT component of the transgene prevented expression of the VP portion in neuronal cells, while sequences within VP-B repressed the hypothalamic expression of bOT3.5. Support for the concept of cross-talk between the VP and OT genes has also come from the experiments of Young et al (37). These authors described mice bearing a minilocus transgene (V2) consisting of construct ROT1.63 (see above and Fig.…”

“…bOT6.4 differed from bOT only in that the sequence upstream of the start of transcription consisted of 3.0 kbp, rather than the 0.6 kbp found in the latter transgene. Young et al (37) have described a similar situation with a rat OT transgene. No transgenic mice were represented in the pups resulting from the injection of fertilized one-cell mouse eggs with ROT1.63, which consisted of the entire rat OT structural gene flanked by 0.36 kbp of upstream and ϳ0.4 kbp of downstream sequences (Fig.…”

“…The VP component of the V2 minilocus was again silent. However, the OT transcription unit present in V2 was expressed in oxytocinergic magnocellular neurons (37)(38)(39). Thus, elements within the VP component must repress the deleterious effects of the OT portion, allowing transgenic mice to be generated.…”

Transgenic mouse models of vasopressin expression.

“…Some animals carrying this construct also had low-level expression in hypothalamus. 48 Young et al 49 developed transgenic mice using a chimeric gene containing the rat vasopressin coding sequence with 1.4 kb of upstream sequence and 0.3 kb of downstream sequence fused to the rat oxytocin gene with 0.4 kb of upstream sequence. Rat oxytocin mRNA was detected in the oxytocin-expressing magnocellular neurons of the hypothalamus, but no transgenic rat vasopressin mRNA was detected in the brain.…”