The renin-angiotensin system plays a major role in the regulation of blood pressure and electrolyte homeostasis in mammals. In this study, we subjected transgenic mice containing a human renin genomic construct to a variety of pharmacological and physiological manipulations to test whether expression of the human renin gene and release of active human renin is appropriately regulated in this model. These manipulations were designed to test major regulators of renin release, including angiotensin II, the macula densa, renal perfusion pressure, and β-adrenergic receptors. We used human plasma renin concentration and human renal renin mRNA levels to document the response of the transgene to these stimuli. Human plasma renin concentration increased in response to both angiotensin-converting enzyme inhibition with captopril and isoproterenol and decreased after a high salt diet. A low salt or sodium-deficient diet did not stimulate renin release. Human renin mRNA levels in kidney increased after captopril but were unchanged in the other experimental groups. We also measured the levels of human renin mRNA in double transgenic mice containing the same human renin gene in addition to the human angiotensinogen gene. These mice are chronically hypertensive and have increased circulating levels of angiotensin II. Human renin mRNA levels in the kidney were paradoxically elevated compared with their single transgenic normotensive counterparts. These transgenic mice provide a model for examination of human renin regulation and may help elucidate the molecular mechanisms that regulate the gene in response to physiological cues.
The renin-angiotensin system plays an integral role in BP regulation and electrolyte homeostasis in mammals. It has also been hypothesized to aid in the regulation of local tissue and organ blood flows in an autocrine/paracrine fashion and to play a role in renal development. The genes of the renin-angiotensin system have also been linked to or associated with hypertension in several animal models and in humans.1 2 Renin, an aspartyl protease, initiates a series of proteolytic reactions that lead to the production of Ang II, the biologically active peptide of the system. Although the regulation of renin expression in rodents is well characterized, until recently, the physiological responses controlling hRen gene regulation have largely gone unexplored because of a lack of human tissues and adequate cell lines. Stable transfections of existing non–renin-expressing cell lines with an hRen gene construct have given information concerning intracellular processing and secretion of renin,3 but since these cells do not secrete endogenously produced renin, one cannot state with certainty whether these cells contain all the factors necessary to correctly regulate renin gene expression. The recent description of a human pulmonary carcinoma cell line, Calu-6, that does endogenously secrete renin may provide a system for the study of renin gene regulation in a cell-culture environment.4 Nevertheless, an in vivo system would provide a more ideal model for the study of renin gene regulation, and the creation of transgenic animals containing the hRen gene has provided these models. In both transgenic mouse and transgenic rat models, expression of hRen mRNA has been shown to be appropriately tissue specific and cell specific.5 6 7 8 In addition, baseline physiological regulation has been examined, such as the response to sodium depletion9 or angiotensin-converting enzyme inhibition,5 10 but an extensive investigation of gene regulation in transgenic animals has not been performed. Renin gene expression and protein secretion, for example, are negatively regulated by the Ang II feedback loop, increases in renal perfusion pressure, systemic arterial pressure, and plasma sodium sensed by the macula densa.11 On the other hand, renin gene expression and secretion are augmented by increases in renal nerve activity, decreases in plasma sodium, and activation of β-adrenergic receptors. Moreover, if we are to use transgenic models to further our understanding of the regulation of renin gene expression and the genetics of hypertension, it becomes critically important to determine whether the transgenes respond to known physiological and pharmacological stimuli. Therefore, the purpose of this study was to examine the regulation of hRen mRNA and active hRen secretion in response to a variety of physiological and pharmacological stimuli in transgenic mice that contain the hRen gene alone or in double transgenic hypertensive mice containing both the hRen and hAng genes.
Creation of Transgenic Mice, Mouse Care, and Animal Husbandry
Both hRen and hAng transgenic mice were generated and characterized as previously described.5 12 Double transgenic mice were created by breeding heterozygous hRen transgenic mice with heterozygous hAng transgenic mice.13 Twenty-five percent of the offspring were doubly transgenic. hRen transgenic line 9 and hAng transgenic line 204/1 were used in the generation of double transgenic mice. Positive transgenic mice were differentiated from nontransgenic littermates through polymerase chain reaction of genomic DNA purified from tail biopsy samples using species-specific hRen and hAng primers as previously described.5 12
All mice were fed standard mouse chow and water ad libitum unless otherwise indicated by the experimental protocol. Mouse care met or exceeded the standards set forth by the National Institutes of Health in the Guidelines for the Care and Use of Experimental Animals. Procedures were approved by the University Animal Care and Use Committee at the University of Iowa.
Mice were killed by CO2 asphyxiation. Approximately 0.5 mL of fresh blood was obtained from the aorta and placed in chilled tubes containing 2.5 μL of 0.5 mol/L EDTA. The specimen was immediately spun in a prechilled centrifuge at 14 000 rpm for 5 minutes, and a 150-μL plasma sample was obtained and immediately frozen at −80°C until radioimmunoassay. Kidney samples were obtained immediately after death, were flash-frozen on dry ice, and were stored at −80°C. All perirenal fat and capsular tissue were removed before freezing.
Transgenic mice and nontransgenic control littermates were placed in the following experimental protocol groups:
A. No treatment: Mice in this group (n=10 hRen line 9, n=17 hRen line 10, n=7 controls) received standard mouse chow and water ad libitum with no additional treatments or measures before death. Normal chow contained 0.31% sodium and 0.48% chloride (Teklad LM-485).
B. Captopril treatment: Captopril was dissolved in the drinking water (0.5 mg/mL) to give approximately 100 mg/kg per day of captopril. Treatment was for 10 days (n=5 hRen line 10, n=5 controls).
C. Captopril/low salt treatment: Captopril was dissolved in the drinking water to give approximately 100 mg/kg per day of captopril.5 In addition, mice were fed a low salt diet (Teklad No. 7034) containing 0.08% sodium and 0.13% chloride. Treatment was for 10 days (n=9 hRen line 9, n=6 hRen line 10, n=6 controls).
D. Low salt diet: Mice received a low salt diet alone (Teklad No. 7034) for 10 days (n=5 hRen line 9, n=4 hRen line 10, n=3 controls).9
E. High salt diet: Mice received a high salt diet (Teklad No. 7033) containing 3.18% sodium and 4.91% chloride for 10 days (n=4 hRen line 9, n=7 hRen line 10, n=4 controls).
F. No salt diet: Mice received a sodium-deficient diet (Teklad TD 90228) containing 0.01% sodium and 0.05% chloride for 10 days (n=6 hRen line 10, n=2 controls).
G. No salt diet plus furosemide: Mice received a sodium-deficient diet (Teklad TD 90228) for 10 days and 20 mg/kg furosemide IP on days 3, 5, and 10 of treatment (n=8 hRen line 10, n=7 controls).14 15
H. Isoproterenol: Mice received 250 μg/kg isoproterenol SC 15 minutes before death (n=3 hRen line 9, n=6 hRen line 10, n=3 controls).16
I. Isoproterenol/propranolol: Mice received 10 mg/kg propranolol SC 5 minutes before 250 μg/kg isoproterenol SC.17 Mice were killed 15 minutes after isoproterenol injection (n=4 hRen line 10, n=2 controls).
J. Response to chronic hypertension: Double transgenic mice containing both the hRen and hAng transgenes (hRen line 9×hAng line 204/1, n=8) along with hRen line 9 control mice (n=8) were killed and their tissues studied. Both groups received normal chow.
We performed PRA and PRC assays with a RIANEN Ang I 125I radioimmunoassay kit (DuPont) following the directions and using reagents supplied by the manufacturer. The kit was designed to provide optimal pH for conversion of angiotensinogen to Ang I and to inhibit angiotensinases. Previous work in our laboratory demonstrated that Ang I levels remained stable for several hours of incubation at 37°C, demonstrating the absence of angiotensinases in the samples.12 All samples were frozen and thawed once. For the assay, plasma samples were thawed in an ice bath. Once thawed, 3 μL dimercaprol, 3 μL 8-hydroxyquinoline, and 300 μL maleate buffer were added to the plasma (all reagents were obtained with the radioimmunoassay kit). Samples were then split into four tubes. Tube A was incubated at 4°C with no further additives. Tube B was incubated at 4°C with the addition of 1 μg purified hAng (Scripps Laboratories). Tube C was incubated at 37°C with no additional additives. Tube D was incubated at 37°C with the addition of 1 μg hAng. Samples were incubated for 1 hour, and then the radioimmunoassay was performed according to the protocol supplied by the manufacturer. Samples were appropriately diluted with reagent blank so that the radioimmunoassay results were on the linear portion of the standard curve. The amount of Ang I generated in each sample was obtained by comparison with a standard curve generated each time the assay was performed. mPRA was obtained by subtracting the amount of Ang I generated per milliliter per hour in tube A (4°C plus no additives) from that in tube C (37°C plus no additives). hPRC was obtained by subtracting the mPRA (Tube C−Tube A) from the value obtained by subtracting the amount of Ang I generated per milliliter per hour in tube B (4°C plus 1 μg hAng) from that in tube D (37°C plus 1 μg hAng): hPRC=[(Tube D−Tube B)−(Tube C−Tube A)]. The amounts of Ang I generated in the 4°C samples (tubes A and B) were routinely between 10% and 15% of the values from the 37°C samples. This reflects either renin activity at 4°C or the endogenous level of Ang I in the plasma samples. Although it would be preferable to compare mPRC with hPRC, mPRA was measured instead for several reasons. First, nephrectomized mouse plasma would need to be used as a substrate for the reaction. Nephrectomizing mice is technically difficult, but it can be performed. Nevertheless, numerous previous studies have shown that nephrectomy in mice increases PRA and results in the release of prorenin from a number of extrarenal sites18 19 ; it is unclear whether the presence of prorenin in the substrate plasma will significantly alter the results. We alternatively considered adding nephrectomized plasma from another species, such as the rat, as a source of angiotensinogen, but species-related differences in the kinetics of the reaction between renin and angiotensinogen was a major concern. Indeed, it has been shown that mRen has increased catalytic activity for rat angiotensinogen.20 Use of plasma from another species, such as sheep, would not allow us to differentiate mRen from hRen. Therefore, mPRA was used to show the Ang I–generating activity of the endogenous mRen gene.
We performed several control experiments to validate our PRA and PRC measurements. First, we demonstrated in a prolonged time course experiment that Ang I levels increased linearly with time (up to 6 hours). This was true when both mRen and hRen activities were measured. These results indicate that we were working with the initial velocity of the renin-angiotensinogen reaction and that the substrates of the reaction, mouse angiotensinogen and hAng, were not in limiting amounts (data not shown). Second, there was no evidence of additional Ang I production in samples from transgenic mice incubated in the presence of either 1.0 or 5.0 μg purified hAng. Third, to verify that the results observed were not artifacts of the blood collection process, we demonstrated that there was no difference in mPRA and hPRC from plasma samples obtained by orbital eye bleed in anesthetized mice compared with plasma samples obtained from the aorta in euthanized mice. There was also no difference in the mPRA and hPRC values from either fresh plasma or once-frozen plasma samples.
For the purposes of definition, PRA measures the total Ang I–generating activity in the plasma and depends on the concentration of circulating renin and angiotensinogen. PRC is a measure of the plasma renin concentration based on Ang I generated in the presence of excess substrate. mPRA in these experiments provided a critical control measure that allowed us to assess the magnitude of responses for the experimental treatments described.
RNase Protection Assay
The expression of hRen mRNA and mouse ren-1c in the kidney was determined with an RNase protection assay. The probes used were a partial hRen cDNA from coordinates 741 to 1148 cloned into pSL301 (Invitrogen), a partial mouse ren-1c cDNA from coordinates 178 to 657 cloned into pCRII (Invitrogen), and a partial mouse β-actin cDNA obtained from the manufacturer (Ambion). Total RNA was isolated from kidney samples by homogenization in guanidinium isothiocyanate followed by phenol emulsion extraction at pH 4.0 using a modification of a method previously described.10 Homogenizations were scaled up to 2.5 mL to increase RNA yield and quality. Samples (10 μg) of total kidney RNA were hybridized to single-stranded labeled antisense RNA probes generated by T3 RNA polymerase with a Maxiscript Kit (Ambion). RNase protection was as described by the manufacturer. Protection products were visualized by electrophoresis through polyacrylamide (5%)/urea (8 mol/L) sequencing gels that were fixed and dried for autoradiography.
Data analysis between different treatment groups was performed by one-way ANOVA, with treatment groups compared with the untreated control group. Individual treatments were also compared pairwise with the control group with the use of an unpaired t test (Student's). We considered a value of P<.05 to be statistically significant. All values are expressed as mean±SE.
Regulation of hRen Release
We measured baseline mPRA and hPRC in two independent lines of transgenic mice to rule out position effects on transgene expression (Fig 1⇓). Endogenous mPRA was similar in both lines (14.2±3.1 ng Ang I/mL per hour for line 9 and 8.6±0.9 for line 10), and mPRA did not differ significantly between transgenic and nontransgenic littermate controls (14.2±3.1 versus 11.1±1.9, respectively, for line 9 and 8.6±0.9 versus 12.4±5.5 for line 10). Transgenic mice had measurable active hRen that was not significantly different between the two lines (10.9±1.2 for line 9 versus 11.6±1.0 for line 10). Essentially no hRen activity was observed in nontransgenic littermates from either line. These results suggest that active hRen is being released into the systemic circulation of these mice.
We examined the regulation of hRen release in treated and untreated mice as described in “Methods.” These results are shown for hRen line 10 in Fig 2⇓ and are summarized along with all PRA data in the Table⇓. hRen PRC was highly elevated in transgenic mice treated with captopril alone and also in mice fed a low salt diet combined with captopril. A low salt diet alone did not elevate hPRC. Further attempts at sodium depletion by feeding mice a no salt diet or a no salt diet plus furosemide did not change hPRC. There was a clear trend toward lower hPRC in high salt–treated mice. Isoproterenol treatment resulted in an elevation in hPRC that was blocked by pretreatment of mice with propranolol. The changes in hPRC in response to the experimental treatments in hRen line 9 largely paralleled those observed in hRen line 10 (Table⇓). There were also no significant differences in mPRA in the untreated, captopril, captopril plus low salt, low salt, no salt plus furosemide, high salt, isoproterenol, and propranolol plus isoproterenol groups when transgenic mice were compared with nontransgenic controls. A small but significant difference in mPRA was noted between no salt–treated transgenic and nontransgenic mice (P<.05).
Regulation of hRen Expression in Kidney
We examined by RNase protection hRen and mRen mRNA levels in the kidney of untreated and chronically treated transgenic and nontransgenic mice (Fig 3⇓). hRen mRNA was clearly evident in the transgenic mice, and no hRen expression was observed in the nontransgenic controls, confirming that the probe was specifically detecting hRen but not mRen mRNA. mRen mRNA was evident in both the transgenic and nontransgenic samples when an mRen mRNA–specific RNase protection probe was used (Fig 3⇓). hRen mRNA levels were significantly increased in captopril-treated mice but were unaltered in the other treatment groups, largely consistent with the hPRC data obtained from the chronically treated mice. mRen mRNA levels were similarly increased in the captopril-treated mice but unaltered in the other treatment groups. We did not examine kidney hRen expression in the acute treatment groups (isoproterenol and propranolol) because we did not expect changes in hRen mRNA levels within the short time frame (15 minutes) of the experiments. Renal hRen mRNA levels were equal in hRen line 9 and hRen line 10, and both lines responded quantitatively equally to the experimental treatments (data not shown).
To assess the hRen mRNA response to elevated BP, we generated double transgenic mice containing both the hRen and hAng genes. These double transgenic mice are chronically hypertensive (mean arterial pressure, 150.6±5.0 mm Hg versus 111.1±5.6 in single transgenic hRen mice)13 and have elevated levels of circulating Ang II (340±58 versus 99.8±13.2 pg/mL). Both elevated arterial pressure and high circulating Ang II should theoretically downregulate renin synthesis and release. Nevertheless, steady-state hRen mRNA was significantly upregulated in double transgenic mice compared with single transgenic controls (Fig 4⇓). Interestingly, mRen mRNA levels were appropriately downregulated by these stimuli (Fig 4⇓).
Regulation of hRen Release and mRNA in Response to Experimental Manipulation
The purpose of this study was to examine the regulation of hRen expression and secretion in response to a variety of physiological and pharmacological manipulations in transgenic mice. Previous studies have shown that transgenic mice containing a genomic construct consisting of all exons and intron sequences and flanked by a relatively small amount of upstream (900 bp) and downstream (400 bp) hRen DNA exhibited a tissue-specific and cell-specific expression pattern largely consistent with the expression of renin genes in rodents.5 6 Since the amount of 5′ flanking DNA used in the construct was rather small, we wished to determine whether the gene was capable of responding to physiological cues that normally regulate renin expression and release.
In the studies performed with the single transgenic mice, release of active hRen was stimulated under conditions of angiotensin-converting enzyme inhibition with captopril or treatment with isoproterenol. The level of steady-state hRen mRNA was also increased in the captopril-treated mice. These changes mirrored the response of the endogenous mRen mRNA and protein to these treatments. It is thought that Ang II participates in a feedback loop that regulates renin release and expression by acting through an angiotensin type 1 receptor–dependent mechanism.21 22 23 24 Although this presumably occurs directly on juxtaglomerular cells, there has not been sufficient evidence reported to prove this directly. On the basis of this model, hRen mRNA and release should increase in the captopril-treated mice because of a relief of Ang II–mediated feedback suppression. mPRA and mRen mRNA were also similarly increased in captopril-treated mice, although the increase in mPRA was not as great as that seen with hPRC. This is most likely because of the fact that angiotensin-converting enzyme inhibition not only decreases circulating Ang II, and increases renin synthesis and release, but also decreases angiotensinogen production and release, which decreases Ang I–generating activity. The hPRC value was unaffected by this because we added excess exogenous hAng to the plasma to determine PRC.
The activation of β-adrenergic receptors by either circulating epinephrine or from the release of norepinephrine from renal sympathetic nerve terminals should stimulate renin release and increase renin mRNA through a cAMP-dependent pathway.25 26 In our experiments, we demonstrated that the circulating level of hRen increased in response to isoproterenol and that the response was specifically receptor mediated by blocking the response with prior administration of the β-adrenergic receptor antagonist propranolol. We did not determine renin mRNA in this experimental group because we did not anticipate that a 15-minute acute treatment would affect the level of steady-state renin mRNA. Moreover, the direct receptor-mediated stimulating effects of chronic isoproterenol treatment on renin mRNA levels would have to be interpreted in the context of its BP-elevating effects. We are currently performing experiments to measure the effect of chronic β-adrenergic receptor stimulation under conditions in which BP is not elevated by coadministration of a vasodilator.
Of all the results presented in this report, the data obtained from the high, low, and no salt treatment groups are the most difficult to reconcile. As expected, mice fed a high salt diet for 10 days exhibited a small but significant decrease in hPRC, presumably because of the activation of macula densa mechanisms.27 28 However, steady-state hRen mRNA in the high salt group did not decrease significantly. Obvious explanations may reflect the short duration of the treatment (10 days) and limitations in detecting a small change in renal hRen mRNA that correlates with only a 30% decrease in plasma levels of active hRen. In contrast to the high salt group, limiting sodium intake by feeding mice low salt or sodium-deficient diets for 10 days had no effect on hPRC or renal hRen mRNA levels. Moreover, there was no additive effect on hPRC levels of diuretic use in mice fed a sodium-deficient diet although a modest increase in mPRA was noted in the transgenic group (Table⇑). In rats, these treatments generally result in a significant increase in PRA and renal renin mRNA. The fact that the mPRA and hPRA levels were coincident throughout these experiments rules out the possibility that the human gene lacks appropriate regulatory elements that respond to signals from the macula densa. Instead, the results suggest that either the magnitude or the duration of the treatments in mice must be significantly increased or that mice are more refractory to changes in dietary sodium. Interestingly, 10 days of high dietary sodium does not elevate BP in several mouse strains (D.C. Merrill and C.D.S., unpublished results, 1995). It will be critically important in future experiments to measure urinary excretion of sodium and plasma sodium to confirm the efficacy of our dietary regimen in modulating plasma sodium levels.
It is also important to note that we cannot rule out extrarenal renin as a source of plasma renin in the transgenic mice. Indeed, hRen is expressed in a variety of tissues besides the kidney, including the lung, reproductive tissues, and adipose tissue.5 Previous studies in rodents and large mammals clearly demonstrate that extrarenal tissues secrete prorenin, and Calu-6 cells, which are derived from human lung, express hRen mRNA endogenously yet secrete only prorenin into the media (D.F. Catanzaro and C.D.S., unpublished results, 1995). Although the submandibular gland can be a source of extrarenal renin in mouse strains that contain the Ren-2 gene, the mice used in our studies were maintained on a C57BL/6 genetic background, which lacks Ren-2 and has 100-fold lower levels of submandibular gland renin mRNA.29 30 Moreover, there is no hRen mRNA in the submandibular gland of line 9 and only low levels in line 10. Submandibular gland–derived renin is unlikely to be responsible for our observations because the magnitude of the responses in the two lines were similar.
Regulation of hRen mRNA in Response to Elevated Ang II and High BP
The most interesting results obtained thus far were found in the comparison of renal renin mRNA levels in hypertensive double transgenic mice and their single hRen transgenic counterparts. Clearly, the expectation was that chronic hypertension would trigger baroreceptor mechanisms and the elevated plasma Ang II levels would trigger the Ang II feedback loop to cause a significant downregulation of renal hRen mRNA in the double transgenic mice. Indeed, we showed that the expression of endogenous mRen mRNA is depressed in the double transgenic mice. These results suggest that the mechanisms that normally are responsible for regulating renin mRNA in response to high BP or high circulating Ang II are intact.
The main question we are left with is, What is the mechanism that is driving elevated hRen mRNA levels in the kidney of the double transgenic mice? First, it is unlikely that expression differences or position effects among lines are responsible because the same hRen line was used for both the single hRen transgenic mice and the double transgenic mice. Interestingly, Lee et al31 observed that expression of both the endogenous rat renin and mRen genes was suppressed in the kidney at baseline in hypertensive TGR(mREN-2)27 transgenic rats containing the mouse Ren-2 gene. Angiotensin-converting enzyme inhibition lifted the suppression of both renin genes and lowered BP, but lowering BP alone without altering Ang II levels did not increase the expression of either renin gene. The authors proposed that locally generated Ang II may be suppressing renin gene expression despite the fact that systemic BP was lowered. These results are consistent with the short feedback regulation of renin by Ang II and support the concept of local tissue generation of Ang II. Nevertheless, local factors modulating the renin gene response cannot explain the dysynchrony in the hRen and mRen gene response observed in our model.
Our results can be explained if the transgene construct used in these experiments does not contain all the appropriate regulatory elements needed to regulate renin gene expression in response to elevated BP and/or increased circulating Ang II. Indeed, the transgene used contains only 896 bp of 5′ flanking DNA. As much as 4.6 kb of 5′ flanking DNA of the mRen promoter is required to target appropriate expression on its own in transgenic mice.32 33 Perhaps the regulatory region controlling the response to these stimuli is located farther upstream. One difficulty with this interpretation is that the level of hRen gene expression significantly increased in response to angiotensin-converting enzyme inhibition. This suggests the possibility that the regulatory elements responsible for controlling the renin gene response to decreased circulating Ang II (as in the captopril-treated mice) are distinctly separate from those controlling the response due to increased BP (or increased Ang II). It is also possible that the increase in renin mRNA due to captopril and the decrease normally caused by baroreceptor mechanisms and the Ang II feedback loop operate by different mechanisms, such as transcriptional versus posttranscriptional mechanisms. The increase in renin mRNA in response to elevated cAMP has been suggested to be partly due to decreased renin mRNA turnover in mouse juxtaglomerular cells34 and human Calu-6 cells.35 We are currently testing some of these hypotheses by generating additional transgenic mice with constructs that contain both increased (5 and 22 kb) and decreased (149 bp) amounts of upstream flanking sequences. We are also experimentally determining whether high circulating levels of Ang II with and without hypertension are responsible for our observations.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|PRA||=||plasma renin activity|
|PRC||=||plasma renin concentration|
Funds to support this work were provided by the National Institutes of Health (NIH) (HL-48058 to C.D.S.), American Heart Association (to C.D.S.), Baxter Healthcare Corp (to C.D.S.), and the Wyeth Pediatric Neonatology Research Fund (to M.W.T.). C.D.S. is an Established Investigator of the American Heart Association. S.B.S. is funded by an NIH training grant (DE00175) and is a member of the Dental Scientist Training Program at the University of Iowa. 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. We would like to thank Norma Sinclair and Lucinda Robbins for technical assistance associated with the generation and characterization of transgenic mice.
- Received September 19, 1995.
- Revision received November 10, 1995.
- Revision received April 8, 1996.
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