EXPRESSION AND FUNCTION OF THE GUANYLIN LIGAND FAMILY

这一应用的总体目标是：1)利用配对配体鸟苷和尿鸟苷的表达模式来了解影响肠上皮细胞转录调控的因素；2)通过建立和研究转基因动物模型来鉴定这些配体的特定生理和病理生理功能。由于鸟苷和尿鸟苷具有明确的空间表达模式，它们在肠道中的高水平表达，以及我们目前拥有的工具，这些成对的基因代表了一个独特的机会来确定基因表达的机制，特别是在结肠和小肠隐窝中。体外技术，包括瞬时转染试验、DNasel超敏反应和足迹技术将被用于指导体内基因表达的研究。表达与荧光素酶报告基因相连的鸟苷和尿鸟苷启动子元件的不同部分的转基因小鼠将被用于定义指导组织特异性表达的序列。为了实现我们的第二个目标，我们将创造一个鸟苷和一个尿鸟苷基因敲除小鼠。我们通过同源重组进行基因打靶的最初努力已经产生了一种围产期致死和/或胚胎致死的表型。为了确定导致这种表型的机制(S)，我们将确定鸟苷和尿鸟苷在发育中的小鼠胚胎、类胚体、胎盘和卵黄囊中的表达部位；为此，我们将使用RT-PCR法、整体贴片法和组织切片原位杂交技术。这将确定鸟苷或尿鸟苷在发育中的小鼠的肠道以及肠外的关键器官中是否表达。基于这些研究的结果和解释，我们将寻求一种策略，通过利用体外或体内条件靶向方法来实现鸟苷素的组织特异性消融。这将产生肠道中缺乏鸟苷和/或尿鸟苷的转基因小鼠。为了明确鸟苷和尿鸟苷在体内的作用，我们将使用生物电、离子通量和原位连接环测量基础和刺激的肠道分泌。根据观察到的鸟苷素的生理作用，我们将研究鸟苷素和尿鸟苷的缺失对小鼠耐盐性、碳酸氢盐缓冲、急性时相损伤、肠运动和肠腺瘤形成的影响。我们将确定鸟苷或尿鸟苷受体在这些小鼠中是否上调，以及鸟苷环化酶C(GC-C)以外的受体是否在任何已发现的表型中发挥作用。如果鸟苷或尿鸟苷失活导致“无基础表型”，我们将寻找允许这种“正常”表型的补偿或冗余机制。

The overall objectives of this application are: 1) to use the expression patterns of the paired ligands guanylin and uroguanylin to understand the factors which contribute to transcriptional regulation in intestinal epithelial cells and 2) to identify the specific physiologic and pathophysiologic functions of these ligands by generating and studying transgenic animal models. Because of the defined spatial patterns of expression of guanylin and uroguanylin, their high levels of intestinal expression, and the tools which we currently possess, these paired genes represent a unique opportunity to identify mechanisms of gene expression, especially in the colon and in the small intestinal crypts. In vitro techniques, including transient transfection assays, DNasel hypersensitivity and footprinting will be used to guide in vivo studies of gene expression. Transgenic mice expressing various portions of the guanylin and uroguanylin promoter elements linked to the luciferase reporter gene will be used to define sequences that direct tissue specific expression. In order to accomplish our second objective we will create a guanylin and a uroguanylin knockout mouse. Our initial efforts at gene targeting by homologous recombination have produced a perinatal-lethal and/or embryo-lethal phenotype. In order to determine the mechanism(s) responsible for this phenotype, we will identify the sites of expression of guanylin and uroguanylin in the developing mouse embryo, embryoid bodies, placenta and yolk sac; to do this we will use RT-PCR, whole mount and tissue slice in situ hybridization. This will identify whether guanylin or uroguanylin is expressed in the developing intestine as well as in critical organs outside the intestine in the developing mouse. Based on the results and interpretation of these studies, we will pursue a strategy to effect tissue-specific ablation of guanylin by taking advantage of an in vitro or an in vivo conditional targeting approach. This will generate transgenic mice lacking guanylin and/or uroguanylin in the intestine. To discern the role of guanylin and uroguanylin in vivo, we will measure basal and stimulated intestinal secretion using bioelectric, ion flux and in situ ligated loop measurements. Based on the observed physiologic actions of guanylin, we will investigate the effect of guanylin and uroguanylin loss on salt tolerance, bicarbonate buffering, an acute phase injury, intestinal motility and intestinal adenoma formation. We will determine whether guanylin or uroguanylin receptors are upregulated in these mice and whether receptors other than guanylyl cyclase C (GC-C), play a role in any identified phenotype. Should guanylin or uroguanylin inactivation result in "no basal phenotype," we will search for compensatory or redundant mechanisms which permit this "normal" phenotype.