Fields Raymond L, House Shirley B, Gainer Harold
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurosci. 2003 Aug 27;23(21):7801-9. doi: 10.1523/JNEUROSCI.23-21-07801.2003.
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.
以往利用转基因小鼠和大鼠模型对下丘脑催产素（OT）和加压素（VP）细胞特异性基因表达的研究，将注意力集中在基因间区域（IGR），认为其是关键增强子元件所在位点。在本研究中，我们使用大鼠下丘脑的器官型切片培养物作为体外模型，并采用粒子介导的基因转移（生物弹道学）转染方法，以确定OT和VP基因之间IGR中负责下丘脑特异性基因表达的关键DNA序列。将小鼠VP基因的5'侧翼区域从3.5 kbp减少到288 bp，并未改变其在下丘脑切片中的表达效率。所有后续的VP构建体均基于此288 bp的VP基因构建体，仅对IGR进行了改变。这些研究使用了各种构建体，其中OT和VP启动子驱动增强型绿色荧光蛋白报告基因表达，结果表明IGR对于体外下丘脑切片中OT和VP基因的表达是必需的。IGR中负责OT和VP基因表达的DNA序列位于VP基因第3外显子下游紧邻的178 bp区域内。此外，IGR中的另一个区域，即OT基因第3外显子下游紧邻的430 bp区域，包含一个下丘脑OT基因表达的正调控元件。178 bp和430 bp区域内DNA序列的比对揭示了四个共同序列（基序），它们可能是IGR中调节OT和VP基因下丘脑特异性表达的假定增强子的候选序列。
