In vivo expression profile of a H+-K+-ATPase alpha2-subunit promoter-reporter transgene.
In vivo expression profile of a H+-K+-ATPase alpha2-subunit promoter-reporter transgene.
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H -K -ATPase α2 亚基启动子-报告基因转基因的体内表达谱。
DOI：
10.1152/ajprenal.00043.2003
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Kone,BruceC
中科院分区：
文献类型：
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作者：
Zhang,Wenzheng;Xia,Xuefeng;Zou,Lei;Xu,Xiangyang;LeSage,GeneD;Kone,BruceC
由于对小鼠H ~+-K ~+-ATP酶α2（HKα2）基因或其他表达部分局限于集合管的基因的转录调控的分子基础知之甚少，特别是在体内，我们开发了携带插入型HKα 2启动子-报告基因构建体的转基因小鼠。在这些小鼠中，HKα25′侧翼区的− 7，264/+253区域控制报告基因增强型绿色荧光蛋白（EGFP）的表达。用荧光显微镜和免疫印迹法检测HKα2/EGFP转基因细胞的表达模式。在10个主要器官检查，EGFP免疫反应性检测丰富的肾脏，在很小的程度上，在脑和肺。在肾脏内，EGFP荧光仅在转基因小鼠的集合管中检测到，并与内源性HKα 2和水通道蛋白-2的细胞分布共定位，与已知的内源性HKα 2在主细胞中的表达模式一致。令人惊讶的是，通过免疫印迹或荧光显微镜在远端结肠中没有明显的转基因表达，而远端结肠是内源性HKα 2表达最高的部位。虽然先前的稳态mRNA水平研究表明肾脏和结肠中HKα 2基因的调控存在差异，但我们的结果首次提供了HKα 2基因在这些器官中的差异转录控制的直接证据，并表明5′-侧翼区以外的区域或其他调控因子在远端结肠中的HKα 2表达中起作用。
Because little is known about the molecular basis of transcriptional regulation of the murine H+-K+-ATPase α2(HKα2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HKα2promoter-reporter gene construct. In these mice, the region −7,264/+253 of the HKα25′-flanking region controls expression of the reporter gene enhanced green fluorescent protein (EGFP). Patterns of HKα2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of 10 major organs examined, EGFP immunoreactivity was detected abundantly in the kidney, and to a far lesser extent, in the brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of transgenic mice and colocalized with the cellular distribution of both endogenous HKα2and aquaporin-2, consistent with the known expression pattern of endogenous HKα2in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of the highest endogenous HKα2expression. Although previous studies of steady-state mRNA levels suggested differences in HKα2gene regulation in the kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HKα2gene in these organs and suggest that regions outside the 5′-flanking region or other regulatory factors play a role in HKα2expression in the distal colon.