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METHODS IN CELL PHYSIOLOGY

V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice

R. Lance Miller,¹ Ping Zhang,¹ Maren Smith,¹ Valerie Beaulieu,² Teodor G. Punescu,² Dennis Brown,^2,3 Sylvie Breton,^2,3 and Raoul D. Nelson¹

¹Department of Pediatrics, University of Utah, Salt Lake City, Utah; ²Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown; and ³Department of Medicine, Harvard Medical School, Boston, Massachusetts

Submitted 10 February 2004 ; accepted in final form 13 December 2004

The kidney, epididymis, and lungs are complex organs with considerable epithelial cell heterogeneity. This has limited the characterization of pathophysiological transport processes that are specific for each cell type in these epithelia. The purpose of the present study was to develop new tools to study cell-specific gene and protein expression in such complex tissues and organs. We report the production of a transgenic mouse that expresses enhanced green fluorescent protein (EGFP) in a subset of epithelial cells that express the B1 subunit of vacuolar H⁺-ATPase (V-ATPase) and are actively involved in proton transport. A 6.5-kb portion of the V-ATPase B1 promoter was used to drive expression of EGFP. In two founders, quantitative real-time RT-PCR demonstrated expression of EGFP in kidney, epididymis, and lung. Immunofluorescence labeling using antibodies against the B1 and E subunits of V-ATPase and against carbonic anhydrase type II (CAII) revealed specific EGFP expression in all renal type A and type B intercalated cells, some renal connecting tubule cells, all epididymal narrow and clear cells, and some nonciliated airway epithelial cells. No EGFP expression was detected in collecting duct principal cells (identified using an anti-AQP2 antibody) or epididymal principal cells (negative for V-ATPase or CAII). This EGFP-expressing mouse model should prove useful in future studies of gene and protein expression and their physiological and/or developmental regulation in distinct cell types that can now be separated using fluorescence-assisted microdissection, fluorescence-activated cell sorting, and laser capture microdissection.

collecting duct; enhanced green fluorescent protein

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