Abstract
Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression of HAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice.
Highlights
Transgenic mice were generated containing a 1542base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of targeting inducible expression of renin-angiotensin system components to the kidney
We chose the KAP promoter for these studies by virtue of its kidney-specific expression and because, like angiotensinogen, KAP is expressed in proximal convoluted tubule (PCT) cells and its expression is responsive to androgens
Transgenic mice were generated with a construct containing 1542 bp of the KAP promoter fused to a 10.3-kb HAGT genomic clone encompassing exons II, III, IV, and V, the intervening introns, a 70-bp segment derived from the 3Ј-end of intron I, and the native 3Ј-end of the HAGT gene containing the poly(A) sites (Fig. 1A)
Summary
Transgenic mice were generated containing a 1542base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of targeting inducible expression of renin-angiotensin system components to the kidney. There was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice. In situ hybridization studies showed that KAP mRNA is localized in PCT cells of the renal cortex in normal male and testosterone-induced female mice [27]; and its expression is under complex hormonal control involving androgens, estrogens, and pituitary hormones [28, 29]. We show that under the control of 1542 bp of the KAP promoter, HAGT gene expression is restricted to renal PCT cells and is strongly induced in female mice in response to androgen treatment
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