Fenton Robert A, Shodeinde Adetola, Knepper Mark A
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Am J Physiol Renal Physiol. 2006 Jan;290(1):F188-95. doi: 10.1152/ajprenal.00285.2005. Epub 2005 Aug 9.
The urea transporters, UT-A1 and UT-A3, two members of the UT-A gene family, are localized to the terminal portion of the inner medullary collecting duct (IMCD). In this manuscript, we demonstrate that 4.2 kb of the 5'-flanking region of the UT-A gene (UT-Aalpha promoter) is sufficient to drive the IMCD-specific expression of a heterologous reporter gene, beta-galactosidase (beta-Gal), in transgenic mice. RT-PCR, immunoblotting, and immunohistochemistry demonstrate that within the kidney, transgene expression is confined to the terminal portion of the IMCD. Colocalization studies with aquaporin-2 show that expression is localized to the principal cells of the IMCD2 and IMCD3 regions. Utilizing beta-Gal activity assays, we further show that within the kidney, the beta-Gal transgene can be regulated by both water restriction and glucocorticoids, similar to the regulation of the endogenous UT-A gene. These results demonstrate that 4.2 kb of the UT-Aalpha promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific and cell-specific fashion in transgenic mice.
尿素转运蛋白UT-A1和UT-A3是UT-A基因家族的两个成员，定位于髓质内集合管（IMCD）的末端部分。在本论文中，我们证明UT-A基因5'侧翼区域的4.2 kb（UT-Aα启动子）足以驱动转基因小鼠中异源报告基因β-半乳糖苷酶（β-Gal）的IMCD特异性表达。逆转录聚合酶链反应（RT-PCR）、免疫印迹和免疫组织化学表明，在肾脏内，转基因表达局限于IMCD的末端部分。与水通道蛋白-2的共定位研究表明，表达定位于IMCD2和IMCD3区域的主细胞。利用β-Gal活性测定，我们进一步表明，在肾脏内，β-Gal转基因可受限水和糖皮质激素调节，类似于内源性UT-A基因的调节。这些结果表明，4.2 kb的UT-Aα启动子足以在转基因小鼠中以组织特异性和细胞特异性方式驱动异源报告基因的表达。
