ANDROGEN CONTROL OF TARGETED GENE EXPRESSION IN KIDNEY

本研究的目标是建立一个模型系统， 转基因小鼠中的异位基因表达可以靶向 肾近曲小管细胞受条件性 调控启动子 该提议是基于以前的实验 主要来自这个实验室， Kap，肾雄激素调节蛋白（KAP）基因， 小鼠肾脏。 Kap仅在雄性肾脏中表达， 是由雄激素引起的 然而，基因在 多激素控制，为了建立雄激素调节 转基因的表达，我们建议检查启动子 参与Kap激素调节的元素。 的目标 这些实验是为了确定最小的雄激素调节， 启动子来驱动转基因的表达。 主要是在体内 将采取的办法，以确定和定性的 调节Kap对激素应答的启动子元件 刺激. 将在培养细胞中进行启动子分析， 确认和完善体内获得的数据。 为了实现目标， 该项目将实现以下具体目标。 第一、 Kap启动子功能将通过以下作图在体内检查： DNA酶I超敏位点并通过体内足迹法鉴定 调节基因的HRE。 第二，转基因小鼠携带 转基因，所述转基因包含融合到多个启动子的不同长度的Kap启动子。 将检测报告基因在细胞中表达该基因的能力。 一种组织特异性的，神经调节的方式来识别最小的 雄激素调节启动子 mRNA的细胞定位和 转基因和内源性Kap基因的蛋白产物 将被确定。 第三，将Kap启动子与基因融合 SV40 T抗原靶向肾近曲小管肿瘤发生 细胞 所产生的肿瘤将被表征和分散作为一个来源 用于衍生表现出所选择的细胞的稳定细胞系， 表型，即，雄激素调节转基因的表达。 第四，永生化细胞将用于建立体外 转染系统，其中来自早期体内实验的数据将 得到确认和完善。 这些细胞的核提取物将提供 介导启动子调节的蛋白质来源。 最后，将对KAP进行描述，以便于识别和 克隆该基因的人类同源物。 严格的规定 该基因的突变表明KAP在肾脏中的重要作用， 为肾近曲小管提供第一基因靶向能力。

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.