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绿色荧光蛋白在转基因小鼠肾主细胞中的特异性表达。

Renal principal cell-specific expression of green fluorescent protein in transgenic mice.

作者信息

Zharkikh Ludmilla, Zhu Xiaohong, Stricklett Peter K, Kohan Donald E, Chipman Greg, Breton Sylvie, Brown Dennis, Nelson Raoul D

机构信息

Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132, USA.

出版信息

Am J Physiol Renal Physiol. 2002 Dec;283(6):F1351-64. doi: 10.1152/ajprenal.0224.2001.

Abstract

The purpose of this study is to develop transgenic mice with principal cell-specific expression of green fluorescent protein (GFP). After the cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of the promoter were used to drive expression of GFP in transgenic mice. In transgenic mice, GFP was selectively expressed in principal cells of the renal collecting duct and not in intercalated cells. Expression was increased by dehydration of mice. AQP2 and GFP expression was maintained in primary cultures of renal medulla that were stimulated with cAMP or vasopressin analogs. GFP-expressing cells were then isolated by fluorescence-activated cell sorting. RT-PCR analysis showed expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and cAMP response element binding protein but not H+-ATPase B1 subunit or anion exchanger 1. After expansion of these cells in culture, RT-PCR analysis showed continued expression of the same genes. This pattern of gene expression is that of principal cells rather than intercalated cells. This transgenic mouse model can be used in future studies of gene expression during the development, differentiation, and maturation of renal principal cells.

摘要

本研究的目的是培育出绿色荧光蛋白(GFP)在主细胞中特异性表达的转基因小鼠。在对小鼠水通道蛋白-2(AQP2)基因进行克隆和测序后,用9.5 kb的启动子驱动GFP在转基因小鼠中表达。在转基因小鼠中,GFP在肾集合管的主细胞中选择性表达,而在闰细胞中不表达。小鼠脱水后表达增加。在经环磷酸腺苷(cAMP)或加压素类似物刺激的肾髓质原代培养物中,AQP2和GFP表达得以维持。然后通过荧光激活细胞分选分离出表达GFP的细胞。逆转录-聚合酶链反应(RT-PCR)分析显示有水通道蛋白2、水通道蛋白3、水通道蛋白4、加压素2型受体和cAMP反应元件结合蛋白的表达,但没有H⁺-ATP酶B1亚基或阴离子交换蛋白1的表达。在培养中扩增这些细胞后,RT-PCR分析显示相同基因持续表达。这种基因表达模式是主细胞而非闰细胞的模式。这种转基因小鼠模型可用于未来肾主细胞发育、分化和成熟过程中基因表达的研究。

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