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The Journal of Neuroscience, August 27, 2003, 23(21):7801-7809

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Regulatory Domains in the Intergenic Region of the Oxytocin and Vasopressin Genes that Control their Hypothalamus-Specific Expression In Vitro

Raymond L. Fields, Shirley B. House, and Harold Gainer

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Previous studies of oxytocin (OT) and vasopressin (VP) cell-specific gene expression in the hypothalamus using transgenic mouse and rat models focused attention on the intergenic region (IGR) as the site of critical enhancer elements. In this study, we used organotypic slice-explant cultures of rat hypothalamus as in vitro models, and particle-mediated gene transfer (biolistics) transfection methods to identify critical DNA sequences in the IGR between the OT and VP genes responsible for hypothalamic-specific gene expression. Reducing the 5' flanking region in the mouse VP gene from 3.5 kbp to 288 bp did not alter the efficacy of its expression in hypothalamic slices. All subsequent VP constructs were based on this 288 bp VP gene construct with changes made only to the IGR. These studies, which used various constructs with OT and VP promoters driving enhanced green fluorescent protein reporter gene expression, demonstrated that the IGR is necessary for OT and VP gene expression in hypothalamic slices in vitro. The DNA sequences in the IGR responsible for both OT and VP gene expression were located in a 178 bp domain immediately downstream of exon 3 of the VP gene. In addition, another domain in the IGR, 430 bp immediately downstream of exon 3 of the OT gene, contained a positive regulatory element for OT gene expression in the hypothalamus. Alignment of the DNA sequences in the 178 and 430 bp domains reveals four common sequences (motifs) that may be candidates for the putative enhancers in the IGR that regulate OT and VP gene hypothalamic-specific expression.

Key words: hypothalamus; organotypic culture; biolistics; gene expression; vasopressin; oxytocin

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Received March 24, 2003; revised June 12, 2003; accepted June 16, 2003.

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