Abstract
BackgroundUromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.ResultsThe GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney.ConclusionThe goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.
Highlights
Uromodulin is the most abundant protein found in the urine of mammals
It is an 85-kD glycosylphosphati-dylinositol (GPI)-anchored glycoprotein secreted from the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in kidney [1]
Cloning and characterization of the goat uromodulin gene promoter and its partial 3' end A 3.7 kb fragment of the goat uromodulin gene fragment containing a 1.5 kb 5' flanking region, exon 1, intron 1, exon 2 and part of intron 2 was cloned by PCR genomic walking based on the bovine sequence [7]
Summary
Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. Uromodulin is the most abundant protein in the urine of all placental mammals, with approximately 50–200 mg released per day. It is an 85-kD glycosylphosphati-dylinositol (GPI)-anchored glycoprotein secreted from the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in kidney [1]. Physiological functions of uromodulin have remained elusive, but recent knock-out studies have suggested that it plays a role in defense against urinary tract infection [3,4]. It may have an immuno-suppressive role [5]. The abundance of the uromodulin protein in urine makes the uromodulin promoter a good candidate for driving the production of recombinant (rc)-
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