Gongyu Yang and Curt D. Sigmund Are Dispensable in Transgenic Mice Regulatory Elements Required for Human Angiotensinogen Expression in HepG2 Cells

Previous researchers have identified two sequences present upstream (angiotensinogen gene–activating element [AGE2]) and downstream (d61–2) of the human angiotensinogen gene that act as cell-specific enhancers of transcription in transiently transfected HepG2 cells. To examine the importance of these two sequences in regulating tissueand cell-specific expression of the gene in vivo, we generated transgenic mice containing the mutations in the context of a genomic transgene previously shown to exhibit appropriate tissue and cell specificity. The ability of these sequences to enhance transcription of a basal human angiotensinogen promoter was confirmed in transient transfection assays in HepG2 cells, and mutations within the AGE2 and d61–2 sequences abolished transactivation of the promoter. Tissueand cell-specific expression was examined in three lines of transgenic mice carrying the d61–2 mutation, two lines of transgenic mice carrying the AGE2 mutation, and three founder transgenic mice carrying a double-mutant construct. Although the absolute levels of expression varied among lines, the pattern of tissue-specific expression was essentially unaltered by the mutations. In situ hybridization confirmed that the mutations were also dispensable for proximal tubule-specific expression within the kidney. Finally, a comparison of transgene expression with transgene copy number revealed a direct proportionality in liver (R5.77, P5.0014) and kidney (R5.76, P5.0024). These results clearly demonstrate that these sites, which strongly induce promoter activity in cells in culture, are not required for appropriate expression of the gene when present in a genomic construct in vivo. (Hypertension. 1998;31:734-740.)