Departments of Internal Medicine and Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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In the kidney, renin gene expression is exquisitely localized to the juxtaglomerular (JG) cells lining the afferent arteriole, having the capacity to regulate renin synthesis in response to a variety of physiological cues. We investigated human renin gene expression in transgenic mice containing a genomic construct driven by 149 bp of its proximal promoter to elucidate whether this was sufficient to confer JG-specific expression. Whereas human renin mRNA was permissively expressed in most tissues, the transgene was expressed mainly in JG cells in the kidney. Active human renin and human prorenin were found in the systemic circulation at levels consistent with previous transgenic models. Remarkably, two lines displayed an appropriate upregulation of transgene mRNA in response to angiotensin-converting enzyme inhibition, and two lines exhibited a downregulation of transgene mRNA in response to subpressor and pressor doses of ANG II. Our results suggest that 149 bp of the human renin proximal promoter, in a context of a genomic construct, are sufficient to confer human renin expression in renal JG cells and at least some aspects of appropriate regulation.
angiotensin II; captopril; blood pressure; gene regulation; juxtaglomerular cells
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INTRODUCTION |
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THE RENIN-ANGIOTENSIN system has long been known to be
involved in physiological processes regulating blood pressure and
electrolyte homeostasis. In most mammals, renin is thought to be the
rate-limiting regulator of the enzymatic cascade leading to the
production of ANG II. Renin is produced and processed in renal
juxtaglomerular (JG) cells, which are primarily responsible for
synthesis of blood-borne renin. Transcription of the renin gene and
release of renin from these cells are stimulated in response to a
variety of physiological and molecular signals resulting from reduced
renal perfusion pressure, sodium depletion, and -adrenergic traffic
from the adrenal gland and sympathetic nerves (11). On the contrary,
renin protein release and mRNA production is thought to be
downregulated under conditions of elevated pressure or in response to
ANG II-mediated feedback (12). In the end, activation of angiotensin
receptors results in a wide array of systemic effects, such as
vasoconstriction, and tissue-specific effects, such as aldosterone
release, enhancement of renal sodium transport, regulation of renal
blood flow, increase in thirst, and facilitation of neurotransmitter
release (reviewed in Refs. 6 and 22). Clearly, maintaining appropriate
regulation of renin production is essential for the maintenance of a
variety of homeostatic processes controlled ultimately by ANG II.
Numerous studies aimed at uncovering the DNA sequences required for signaling proper tissue- and cell-specific expression and physiological regulation of the renin gene have been reported (reviewed in Ref. 28). In an attempt to develop model systems to identify regulatory elements controlling renin transcription in vivo, we previously generated transgenic mice (900-hREN) containing a complete human renin (hREN) genomic sequence spanning a region from 896 bp in the 5' flank (promoter) to 400 bp in the 3' flank of the gene (26). The hREN transgene was expressed in the expected spectrum of tissues, including kidney, adrenal gland, testes, and ovary, and was developmentally regulated (24, 26). Moreover, hREN expression and protein release appropriately increased in response to captopril treatment and decreased in response to high dietary salt (32). Both observations are consistent with the renal JG cell-specific expression of the transgene observed by in situ hybridization (26). However, transgene expression was also evident in a number of extrarenal tissues, some of which are not considered classical renin-expressing sites. In addition, an increase in circulating renin was observed after bilateral nephrectomy (37), and a paradoxical increase in hREN mRNA was observed when hypertensive mice were obtained from breeding 900-hREN transgenic mice with mice expressing human angiotensinogen (hAGT) (32). This suggests that there may be a complex arrangement of regulatory elements controlling both cell-specific expression and regulation in response to physiological cues, and that the 900-hREN transgene may contain some but not all signals required for appropriate regulation.
Other attempts to identify specific regulatory sequences controlling hREN expression by fusing various lengths of the hREN promoter (149, 896, 1244, 3000, and 5000 bp) to reporter genes were unsuccessful. Although we obtained and bred a total of 46 transgenic founders, there was no evidence of significant transgene expression in any construct examined (Sigmund, unpublished observations), suggesting that important regulatory elements may exist either further upstream or within the structural gene itself. Both possibilities are supported by in vitro data (17, 36, 38). Other recent data demonstrating a marked increase in the stability of renin mRNA in response to cAMP treatment in both primary mouse JG cells (4) and hREN expressing Calu-6 cells (27) suggest that fusion transgenes may fail to express in transgenic mice because they lack the hREN mRNA sequence itself.
To address these issues, we have developed new hREN transgenic models that retain their overall genomic structure (all exons and introns) but differ in the amount of 5' flanking sequence present. In the present study, we examined whether a genomic construct containing only a minimal promoter (149 bp) would be sufficient to direct JG cell-specific expression of hREN in transgenic mice. Here we report the surprising finding that, although the transgene was ubiquitously and permissively expressed in most tissues, its expression in kidney was generally restricted to JG cells. Moreover, hREN expression in some transgenic lines was appropriately upregulated after angiotensin-converting enzyme ACE inhibition and downregulated after ANG II infusion.
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METHODS |
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Creation of transgenic mice and animal
husbandry.
The 140-hREN transgene construct was generated by first subcloning the
downstream KpnI to
BglII fragment containing exon
IX (Fig. 1)
from 900-hREN (26) into the pSL301 vector (Invitrogen). The large
KpnI fragment from 900-hREN was then
inserted upstream of the smaller fragment, and the orientation was
verified by sequencing. The 140-hREN construct was prepared for
microinjection by freeing it from the vector by digestion with
NotI and
BglII, followed by gel purification as
previously described (26). Generation of transgenic mice was as
previously described (26). All mice were fed standard mouse chow and
water ad libitum unless otherwise indicated. Care of mice met or
exceeded the standards set forth by the National Research Council in
the Guide for the Care and Use of Laboratory
Animals. All procedures were approved by the University
of Iowa Animal Care and Use Committee. Experimental mice were killed by
CO2 asphyxiation.
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Administration of drugs and blood-pressure
measurements.
Captopril (Sigma) was dissolved in the drinking water (0.5 mg/ml) to
give ~100
mg · kg1 · day
1
of captopril for 10 days, as previously described (26). Microosmotic pumps (Alzet model 1007D) were utilized to administer vehicle (saline),
subpressor (200 ng · kg
1 · min
1),
and pressor (1,000 ng · kg
1 · min
1)
doses of ANG II. Systolic blood pressure was determined with the use of
the Visitech Systems BP-2000 blood pressure monitoring system, an
automated device for measuring systolic blood pressure by tail cuff.
Mice were trained for 7 consecutive days, during which time
blood-pressure measurements were discarded; baseline systolic pressure
was then measured for the next 3 days. The Alzet minipumps were then
implanted under Metofane anesthesia, and measurements were repeated
daily for the next 5 days.
RNase protection assay.
hREN and mouse
ren-1c
mRNA levels were determined with an RNase protection assay (RPA). hREN
and
ren-1c
probes were partial cDNA sequences cloned into pCRII (Invitrogen) and
pGEM-4 (Promega), respectively, and the mouse -actin cDNA probe
template was obtained from Ambion. The
ren-1c
probe was the kind gift of Dr. David C. Merrill (Bowman Gray School of
Medicine). Tissues were homogenized, and RNA purification was conducted
with the use of TriReagent (MRC) and the manufacturer's protocol.
Total RNA (10-20 µg) was hybridized to a cRNA antisense probe
labeled with the use of an in vitro transcription reaction containing
[
-32P]UTP and
purified with the use of a Sephadex G-50 quick spin column (Boehringer
Mannheim). RPAs were performed with the use of a Hyb-Speed RPA kit
(Ambion) and the manufacturer's protocol. Protection products were
visualized by electrophoresis through a 7% polyacrylamide-5 M urea
gel. The length of the full-length probe for hREN, mouse renin (mREN),
and mAct is ~410, 430, and 315 nucleotides, respectively, and the
expected protected fragment size is 300, 326, and 245 nucleotides,
respectively. Quantification of RPAs were performed with the use of a
Molecular Dynamics Storm 620 phosphorimager system and the ImageQuant
software provided by the manufacturer. hREN and mREN mRNA were
normalized for mouse actin expression in each RPA reaction by dividing
the renin mRNA signal by the actin signal. All comparisons for the
captopril and ANG II infusion experiments were run on one to two RPA
gels run simultaneously.
Plasma renin activity and concentration. Plasma renin activity and plasma renin concentration were determined with the use of an ANG I 125I-radioimmunoassay kit (NEN) and purified hAGT substrate (Scripps Laboratories), using the protocol and calculations previously described (32). The trypsin-based prorenin activation protocol was conducted as previously described (23). Briefly, 100 µl of plasma were combined with activation buffer and 75 µg trypsin in a final volume of 300 µl. The reaction was incubated overnight at 4°C with gentle agitation. Activation buffer consisted of 50 mmol/l Tris (pH 7.4), 0.1 mol/l NaCl, 0.5% BSA, 0.1% EDTA, and 0.1% sodium azide.
Immunohistochemistry. Excised kidneys were immediately immersed in 15% dextrose followed by 30% dextrose until they were saturated. Kidneys were then snap frozen on dry ice and sectioned (6- to 14-µm thick). Sections were fixed in 4% paraformaldehyde for 5-10 min. The sections were incubated overnight at 4°C with a polyclonal hREN primary antibody (R12) diluted 1:35 in DMEM with 5% fetal bovine serum. The hREN antibody was a generous gift from Professors Pierre Corvol and Florence Pinet (INSERM U36, College de France, Paris, France) and was described previously (7). The sections were washed with 1× PBS and incubated with an FITC-conjugated anti-rabbit secondary antibody (Pierce) for 30 min at 37°C. After the secondary incubation, slides were rinsed briefly with 1× PBS before being mounted with a cover slip. Sections were examined from five mice from line 2957/1, six mice from line 2814/2, and three mice from 900-hREN line 9 (26). At least eight sections per kidney were examined. The images were captured with a Bio-Rad MRC-1024 Hercules laser scanning confocal microscope equipped with a Kr/Ar laser.
Statistics. All numerical data are presented as means ± SE. Statistical analysis was performed with the use of Student's t-test, using the SigmaPlot or Systat software package.
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RESULTS |
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Transgenic mice were constructed with the hREN gene driven by 149 bp of
native promoter region (Fig. 1). This construct is identical to the
900-hREN transgenic model we previously described except for employing
a shorter promoter (26). Multiple transgenic lines were established,
and the presence of the transgene in each line was confirmed by
Southern blot analysis (Fig. 2). These
results confirmed the presence of an internal 2.0-kb
BamHI fragment (containing exons
III-V; solid arrow) and a larger genomic fragment derived from the
3' end of one transgene (exons VI-IX; hatched arrow) and the
5' end of the next transgene (exons I-II) along the contiguous array made by the insertion of multiple copies of the transgene. The
large fragment allows us to distinguish 900-hREN (open arrow) from
140-hREN (hatched arrow) transgenics at the DNA level.
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An RPA was used to assess the tissue-specific expression of the
transgene. Figure 3 shows a representative
RPA from line 2813/4 probed for hREN (solid arrow) and mouse -actin
(open arrow) as a loading control. High-level expression of the hREN
gene in the lung and adipose tissue is readily apparent after an
overnight exposure (Fig. 3, top), whereas a longer exposure (Fig. 3,
bottom) was required to observe expression in the kidney and other
extrarenal tissues. The results of 16 RPA experiments from an analysis
of six lines of 140-hREN and three lines of 900-hREN mice is shown in
Table 1. Extremely high levels of hREN mRNA
were routinely found in the lung and adipose tissue across all lines of
both constructs. hREN expression was evident in the kidney of five of
six 140-hREN lines examined, and its expression was generally less than
that observed in 900-hREN mice. Expression of the transgene was also
detected in testes, spleen, and skeletal muscle in most of the lines
and was occasionally detected in liver, heart, brain, and salivary
gland. This pattern of expression is reminiscent of the tissue
distribution reported in the 900-hREN animals; however, the expression
of hREN in "ectopic" extrarenal sites was much more pronounced
and variable among lines of 140-hREN mice.
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Immunohistochemistry was conducted with the use of the hREN specific
antibody R12 (7) and an FITC-conjugated anti-rabbit secondary antibody
to determine whether renin expression was properly restricted to renal
JG cells. JG cell localization was evident in the two independent lines
(2814/2 and 2957/1) of 140-hREN mice tested (Fig.
4) and was similarly detected in the kidney
of five different mice from each line. In addition, however, low-level staining was occasionally observed in epithelial and vascular cells in
line 2957/1. The antibody was specific for hREN, as no significant FITC
staining was observed in the nontransgenic control sections. It is
interesting to note that fewer JG apparatuses demonstrated evidence of
hREN expression (judged to be ~10%) in the kidney of 140-hREN mice
than in 900-hREN mice (data not shown). Moreover, captopril treatment
did not significantly increase the number of JG apparatuses expressing
hREN in these mice (data not shown).
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We next assayed for the presence of both active hREN and prorenin in
the plasma, using purified hAGT as the substrate and a radioimmunoassay
designed to measure plasma ANG I. Plasma hREN was detectable in two (of
three) lines tested, at a level comparable with that previously
reported for 900-hREN (Table
2) (32). Moreover, a
majority of the circulating hREN appeared to be in the form of
prorenin. It is likely that expression of hREN in the extrarenal sites
discussed above may contribute a significant amount of prorenin to the
plasma. There was no significant difference in the level of mREN in
either transgenic or nontransgenic control mice.
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Given the JG cell-specific expression described above, we next
investigated whether this transgene responded appropriately to
physiological stimuli that are known to regulate expression of the
renin gene. In the first set of experiments, three lines of 140-hREN
transgenic and nontransgenic mice were treated with the ACE inhibitor
captopril for 10 days. Captopril prevents the conversion of ANG I to
ANG II and thus relieves the negative feedback normally exerted on
renin production, resulting in an increase of renin mRNA. Indeed,
endogenous mREN mRNA was increased approximately fourfold in all three
transgenic lines and nontransgenic littermates (see Fig.
5B). Renal hREN mRNA was
significantly increased in one line (2846/3;
P < 0.02), was increased but did not
reach statistical significance in another line (2957/1;
P = 0.06), and did not increase at all
in third line (2814/2; Fig.
5A). The
increase in hREN mRNA in lines 2846/3 and 2957/1 was approximately
twofold, whereas mREN mRNA increased approximately fourfold. Although
captopril caused an induction in hREN mRNA in kidney, no changes in
hREN mRNA abundance were observed in lung or adipose tissue of line 2846/3 in response to captopril (data not shown).
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In the second set of experiments, two lines of 140-hREN transgenic and
nontransgenic mice were administered either a nonpressor or pressor
dose of ANG II with the use of a subcutaneous microosmotic pump.
Systolic blood pressure was measured in each mouse before and after ANG
II administration to verify the efficacy of each dose of ANG II (Fig.
6). There was no significant difference
between the systolic blood pressure of the saline (vehicle) and
subpressor ANG II infusion groups. However, the pressor dose of ANG II
caused a 25- to 30-mmHg increase in systolic blood pressure that
generally peaked 3 days after implantation of the pump. Endogenous
renal mREN mRNA was significantly downregulated in response to both the
nonpressor (P < 0.04) and pressor
(P < 0.01) dose of ANG II (Fig.
7). Interestingly, a significant decrease
in renal hREN mRNA was observed in line 2814/2 that was similar in
magnitude and significance to endogenous mREN mRNA. As in the captopril experiment, although there was a trend toward reduced hREN mRNA in the
kidney in line 2957/1, it did not reach statistical significance.
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DISCUSSION |
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We report the surprising finding that an hREN genomic construct driven by only a 149-bp minimal promoter is sufficient to target JG cell-specific expression despite permissive expression in most other tissues. Moreover, several lines of mice exhibited appropriate hREN responses to ACE inhibition and ANG II infusion, suggesting that the transgene exhibits at least some aspects of appropriate regulation. When compared with 900-hREN transgenic mice, expression in the kidney was reduced, with fewer JG cells exhibiting renin immunostaining, and expression in ectopic sites was more abundant and variable among transgenic lines.
Among these ectopic sites are lung and adipose tissue, where abundant hREN mRNA was observed. It remains unclear whether the lung and adipose tissue are true ectopic sites. Indeed, low levels of hREN expression have previously been detected in human fetal lung, in tumors derived from the lung, and in Calu-6 cells derived from a human pulmonary carcinoma (16, 30, 31). In addition, hREN mRNA was reported in human adipose tissue and isolated adipocytes (14). It is interesting to note that adipose tissue is a known site of angiotensinogen expression, suggesting the potential for local synthesis of ANG II in fat (14). Moreover, ANG II has been reported to increase prostacyclin synthesis in fat, which can induce the differentiation of preadipocytes to adipocytes and stimulate lipid synthesis and storage (5, 13).
Independent lines were tested for their ability to upregulate and downregulate hREN mRNA in response to ACE inhibition and ANG II treatment, respectively. In lines 2846/3 and 2957/1, captopril caused a 2-fold increase in hREN mRNA, less than the 3.9- and 3.0-fold increases in endogenous mREN mRNA, respectively. As in the captopril experiment, ANG II caused only a modest but not statistically significant decrease in hREN mRNA in line 2957/1. There was no hREN response to captopril in line 2814/2, although mREN mRNA increased 4.3-fold. It is therefore surprising that this same line, which did not respond to captopril, responded appropriately and with the same magnitude as the mREN gene to both nonpressor and pressor doses of ANG II. Moreover, we previously reported that the 900-hREN construct is upregulated both in response to captopril and, paradoxically, in hypertensive hREN/hAGT double transgenic mice (32). We were therefore surprised that two lines of 140-hREN mice exhibited a decrease in hREN mRNA in response to ANG II infusion. Preliminary studies suggest that the mechanism for the upregulation observed in the 900-hREN mice may be due to an increase in pressure and not ANG II per se (H. Keen and C. D. Sigmund, unpublished observation), suggesting the possibility that there is a complicated arrangement of regulatory elements controlling hREN transcriptional responses to physiological cues such as elevated pressure, ANG II, and ACE inhibition. The exact reasons for the discrepant results between constructs remain unclear. It is important to recognize that captopril not only relieves feedback caused by the loss of ANG II but also reduces blood pressure when administered chronically (18). Therefore, the induction of hREN mRNA in the 140-hREN mice in response to captopril may be due to both mechanisms.
Our data suggest that the 140-hREN transgene may have the intrinsic
ability to be appropriately regulated by both ACE inhibition and ANG II
but that other factors may be strongly influencing the response in the
different transgenic lines. The factors influencing regulation of the
transgene by physiological cues may also be effecting variable
expression of hREN in ectopic sites. Of the factors that may be
responsible, transgene position effects are most likely to have the
greatest effect. The site of transgene integration has been repeatedly
documented to have a strong influence on transgene expression, and
these effects will be different among lines because of the utilization
of different insertion sites (1). Transgenes immune to position effects
are generally very large (encoded on BAC or YAC vectors) and contain
dominant control regions that are thought to insulate the transgene
from effects of neighboring DNA or to act as transcriptional enhancers
(10). In our construct, it is likely that the juxtaposition of basal transcription factor-binding sites present in the hREN promoter (Fig.
8) next to anonymous DNA
at or near the insertion site may impart a new or altered set of
transcriptional instructions to the gene. These instructions may
include directing expression to ectopic sites such as the liver, heart,
skeletal muscle, and submandibular gland and may differ from line to
line. Recent results suggest that sequences in the renin promoter
normally interact with an enhancer of transcription located ~2.6 kb
upstream of the mREN gene (21) and 13 kb upstream of the hREN gene
(38). Enhancers of transcription are known to be able to increase
transcription in an orientation- and position-independent manner and
may impart influences on the transgene even if located far upstream or
downstream from the transgene. Therefore, it is possible that the hREN
minimal promoter, which normally responds to signals from the renin
enhancer (which is missing in 140-hREN) may therefore be quite
susceptible to the influence of transcription factors binding near the
integration site.
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Until recently, most studies examining the transcriptional regulation
of the renin gene focused on the renin promoter and employed
transfection of cultured cells. In nearly all studies reported thus
far, the 149-bp promoter, because of the localization of a convenient
KpnI restriction site at 149,
has been considered the basal promoter. Extensions of this sequence
with DNA further 5' were used to identify sequences acting as
enhancers or repressors of transcription (reviewed in
Ref. 28). In vitro data have suggested that the hREN minimal promoter
is extremely weak, driving expression of reporter genes at only a small
fraction of the level of strong viral promoters (17). The 149-bp
promoter contains a TATA box located at
29 and a binding site
homology to the POU-domain transcription factor Pit-1
located at
67. Interestingly, the Pit-1 site has been reported
to be required in conjunction with a cAMP response element (located at
218 and absent from 140-hREN) for the transactivation of the
hREN promoter by cAMP (8, 9, 39). In addition, a search of the 149-bp
promoter revealed potential binding sites for a number of other
transcription factors that are reported to act as transcriptional
activators and repressors in other genes (Fig. 8). It is therefore
tempting to speculate that Pit-1, or some other transcription
factor-binding sites, may be necessary for directing JG cell-specific
expression, although further experimentation will be required before a
specific site can be implicated.
Similar to in vitro studies, in vivo studies initially focused on the
creation of transgenic mice containing a fusion between the hREN
promoter and a reporter gene. Transgenic mice containing constructs
consisting of variable lengths of the hREN promoter (from 149 to
5000) fused to either SV40 T-antigen or luciferase were
generated and either failed to demonstrate any appreciable transgene
expression or required RT-PCR followed by Southern blot for detection
(Sigmund, unpublished observations). These data suggest that the
important regulatory elements may lie either further 5', within
the body of the gene, or in the 3' flanking region. Such a
candidate may be an enhancer of transcription identified upstream of
the mouse and hREN promoters (21, 38). A 4.6-kb segment of the mouse
Ren-2 promoter containing the mREN enhancer confers tissue-specific
expression on an SV40 T-antigen reporter gene (25). Interestingly,
however, a fusion construct consisting of the mouse enhancer upstream
from the hREN promoter (896 bp) and the T-antigen reporter gene failed
to be expressed in transgenic mice (Q. Shi and C.D. Sigmund,
unpublished observation). When these data are taken together, it
suggests that the mechanism controlling tissue- and cell-specific
expression of hREN may be very complicated. That the distance between
the mouse and hREN enhancer sequences and their respective promoters
differ substantially suggests the provocative possibility that the
mechanisms controlling their regulation may be somewhat divergent.
Finally, data supporting a posttranscriptional mechanism regulating renin mRNA stability may further complicate this picture (4, 27). It is tempting to speculate that the process of transcription, controlled by sequences in the 5' flanking region of the gene, in conjunction with mRNA processing signals present at the exon/intron boundaries and signals regulating mRNA stability, located within the mRNA itself, are all intimately coupled. Consequently, disruption of these processes by removal of either 1) regulatory sequences in the promoter through truncation, 2) exons and introns in the hREN structural gene, or 3) the structure of the hREN mRNA may result in loss of renin expression and may explain our previous inability to map regulatory elements controlling expression of hREN in vivo. It is therefore likely that future transgenic studies of the hREN gene will need to incorporate the entire 13-kb exon/intron region of the gene to be appropriately expressed in mice. We are currently examining the expression of two large hREN transgenes (140 kb and 160 kb) encoded on P1 artificial chromosomes to assess whether they are immune to position artifacts and exhibit appropriate regulation.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Pierre Corvol and Florence Pinet for the gift of the R12 hREN antibody and Dr. David Merrill for the gift of a new ren-1c probe. We also acknowledge the excellent technical assistance of Lisa Hancox, Lucy Robbins, and Norma Sinclair for generation of transgenic mice; Debbie Davis for assistance with blood pressure recordings; Haley Williams for assistance with the fluorescence studies; Henry Keen for assistance with implantation of osmotic minipumps; and Lihong Zhao for the Southern blot.
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FOOTNOTES |
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Funds in support of this work were obtained from National Heart, Lung, and Blood Institute Grants HL-55006 and HL-48058 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52617 and the American Heart Association.
C. D. Sigmund was an established investigator of the American Heart Association.
Transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, which is supported in part by the College of Medicine and the Diabetes and Endocrinology Research Center. DNA sequencing was performed at the University of Iowa DNA Core Facility. Confocal microscopy was performed at the University of Iowa Central Microscopy Facility.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. D. Sigmund, Molecular Biology Interdisciplinary Program, Transgenic and Gene Targeting Facility, Dept. of Internal Medicine and Physiology and Biophysics, 2191 Medical Laboratory, The Univ. of Iowa College of Medicine, Iowa City, Iowa 52242 (E-mail: curt-sigmund{at}uiowa.edu).
Received 16 March 1999; accepted in final form 21 May 1999.
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