From the Departments of Internal Medicine (C.D.S.), Physiology and
Biophysics (C.D.S.), and Anatomy (G.Y.), The University of Iowa College of
Medicine (Iowa City).
Correspondence to Curt D. Sigmund, PhD, Director, Transgenic and Gene Targeting Facility, Departments of Internal Medicine and Physiology & Biophysics, The University of Iowa College of Medicine, 6-432 Bowen Science Bldg, Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu
Abstract—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 tissue- and 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. Tissue- and 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
(R=.77, P=.0014) and kidney
(R=.76, P=.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.
© 1998 American Heart Association, Inc.
Scientific Contributions
Regulatory Elements Required for Human Angiotensinogen Expression in HepG2 Cells Are Dispensable in Transgenic Mice
Key Words: mice, transgenic • transfection • gene expression • mutagenesis • in situ hybridization
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