ANDROGEN CONTROL OF TARGETED GENE EXPRESSION IN KIDNEY

这项研究计划的目标是建立一个模型系统 转基因小鼠中的哪些异位基因表达可以作为靶点 有条件控制下的肾近端小管细胞 受调控的启动子。这一提议是基于之前的实验 主要来自这个实验室，这些实验室的特点是 肾脏雄激素调节蛋白(KAP)基因KAP 老鼠肾。KAP仅在男性肾脏中表达， 是由雄激素戏剧性地诱导的。然而，这种基因还没有被发现。 多激素控制，为了建立雄激素调节 转基因的表达，我们建议检测启动子 参与KAP荷尔蒙调节的因素。的目标是 这些实验是为了确定最低限度的雄激素调节 启动子来驱动转基因的表达。A主要在体内 将采取方法来识别和表征 调节KAP对激素反应的启动子元件 刺激。将在培养细胞中进行启动子分析以 确认并改进在体内获得的数据。为实现这些目标 关于该项目，将开展以下具体目标。第一, KAP启动子的功能将通过定位在体内进行检测 DNA酶I超敏部位及活体足迹鉴定 调节该基因的HRE。第二，转基因小鼠携带有 含有不同长度Kap启动子的转基因融合到 报告基因将被检测其表达基因的能力。 一种组织特异性的、激素调节的方式来识别最小的 雄激素调节启动子。信使核糖核酸及其受体的细胞定位 转基因和内源性Kap基因的蛋白质产物 将会被确定。第三，Kap启动子将与基因融合 SV40T抗原靶向肾近端小管的致瘤作用 细胞。由此产生的肿瘤将被表征并作为来源分散 用于获得表现出所选的 表型，即雄激素调节的转基因表达。 第四，永生化细胞将用于建立体外培养的 一种转基因系统，其中来自早期体内实验的数据将 得到确认和提炼。这些细胞的核提取液将提供 调节启动子的蛋白质的来源。 最后，KAP的特征将有助于识别和 克隆该基因的人类同源基因。严格的监管 提示KAP在肾脏和意志中起着重要作用 为肾脏近端小管提供首个基因靶向能力。

The goal of this research proposal is to establish a model system in which ectopic gene expression in transgenic mice can be targeted to renal proximal tubule cells under the control of a conditionally regulated promoter. The proposal is based on previous experiments primarily from this laboratory that have characterized the regulation of Kap, the gene for Kidney Androgen Regulated Protein (KAP) in the mouse kidney. Kap is expressed exclusively in the kidney in males and is dramatically induced by androgen. However, the gene is under multihormonal control and, in order to establish androgen regulated expression of transgenes, we propose to examine the promoter elements involved in the hormonal regulation of Kap. The goal of these experiments is to identify the minimal androgen regulated promoter to drive expression of transgenes. A primarily in vivo approach will be taken to the identification and characterization of promoter elements that regulate the response of Kap to hormonal stimulation. Promoter analysis in cultured cells will be performed to confirm and refine the data obtained in vivo. To accomplish the goals of the project, the following Specific Aims will be undertaken. First, Kap promoter function will be examined in vivo by the mapping of Dnase I hypersensitive sites and by in vivo footprinting to identify HREs that regulate the gene. Second, transgenic mice carrying a transgene comprising various lengths of the Kap promoter fused to a reporter gene will be examined for their ability to express the gene in a tissue-specific, hormonally regulated manner to identify the minimal androgen regulated promoter. The cellular localization of mRNA and protein products of the transgenes and of the endogenous Kap gene will be determined. Third, the Kap promoter will be fused to the gene for SV40 T antigen to target tumorigenesis to renal proximal tubule cells. Resulting tumors will be characterized and dispersed as a source of cells for the derivation of stable cells lines exhibiting the selected phenotype, i.e., androgen regulated expression of the transgene. Fourth, the immortalized cells will be used to establish an in vitro transfection system in which data from earlier in vivo experiments will be confirmed and refined. Nuclear extracts of these cells will provide a source of proteins that mediate the regulation of the promoter. Finally, KAP will be characterized to facilitate the identification and cloning of the human homolog of the gene. The stringent regulation of this gene suggests an important role of KAP in the kidney and will provide the first gene targeting capability to the renal proximal tubule.