INVITED REVIEWNew approaches to genetic manipulation of mice:tissue-specific ex - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：Justin M.Cole, HongXiao, Jonathan W.Adams, Kevin M.Disher, HuiZhao,  Kenneth E.Bernstein作者单位：Department of Pathology, Emory University, Atlanta, Georgia30322 3 A; }8 o1 X8 L) S6 ~7 m' k; }

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      【摘要】5 t1 r$ n) J% _% U$ Z1 v5 `
   Therenin-angiotensin system (RAS) plays a central role in body physiology,controlling blood pressure and blood electrolyte composition. ACE.1(null) mice are null for all expression of angiotensin-convertingenzyme (ACE). These mice have low blood pressure, the inability toconcentrate urine, and a maldevelopment of the kidney. In contrast,ACE.2 (tissue null) mice produce one-third normal plasma ACE but notissue ACE. They also have low blood pressure and cannot concentrateurine, but they have normal indices of renal function. These mice,while very informative, show that the null approach to creatingknockout mice has intrinsic limitations given the many differentphysiological systems that no longer operate in an animal without afunctioning RAS. To investigate the fine control of body physiology bythe RAS, we developed a novel promoter swapping approach to generate amore selective tissue knockout of ACE expression. We used this tocreate ACE.3 (liver ACE) mice that selectively express ACE in the liverbut lack all ACE within the vasculature. Evaluation of these mice showsthat endothelial expression of ACE is not required for blood pressurecontrol or normal renal function. Targeted homologous recombination hasthe power to create new strains of mice expressing the RAS in selectedsubsets of tissues. Not only will these new genetic models be usefulfor studying blood pressure regulation but also they show great promisefor the investigation of the function of the RAS in complicated disease models. 4 T, S# ]2 B8 T, _& B3 l
      【关键词】 angiotensinconverting enzyme blood pressure angiotensin II liver reninangiotensin system
   INTRODUCTION

ROUGHLY ONE-HALF OF ALL AMERICANS diefrom cardiovascular disease. Put differently, more Americans die ofcardiovascular disease than the next seven leading causes of deathcombined ( 1 ). To understand cardiovascular disease, onemust understand how the body regulates blood pressure, a complexprocess governed by many different components, including multiplevasoconstrictors and vasodilators. Among these is the renin-angiotensinsystem (RAS), which produces the vasoconstrictor angiotensin II. Thebasic physiology by which angiotensinogen is degraded to angiotensin IIhas been known for many years, with the importance of this systemunderscored by the enormous clinical utility of pharmaceuticals thatinterrupt angiotensin II formation or action. So with all that is known about the RAS, what could conceivably be new? In fact, the use oftargeted homologous recombination in mouse embryonic stem (ES) cellshas produced a startling series of insights into the operation andfunctional importance of the RAS. This progress has been so rapid anddramatic as to implicate the RAS as easily the most important regulatorof cardiovascular function as measured through blood pressure control.

The use of homologous recombination to modify genes relies on theproperties of cultured ES cells ( 4 ). Originally derived from the inner cell mass of a mouse embryo, these cells arepluripotent; when reinjected back into a mouse blastocyst, the ES cellscontribute to the formation of all the organs of the recipient. Thusthe recipient mouse is chimeric in that its tissues are derived from both wild-type cells and ES cells. Knockout mouse technology depends onhomologous recombination to introduce specific changes into the DNA ofcultured ES cells. Once these cells are used to create a chimericmouse, simple breeding ultimately results in homozygous, heterozygous,and wild-type mice for the modified genetic locus. Although the typicalstrategy is to eliminate a particular gene, knockout technology canproduce virtually any conceivable genetic change, including geneduplication and the selective mutation of amino acid composition.Indeed, as discussed below, the selective modification of a gene (asopposed to its elimination) holds great promise in creating novelgenetic models.
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Similar to all techniques, the use of targeted homologous recombinationin ES cells has both conceptual advantages and disadvantages. Thegreatest advantage is the plasticity of the technology, allowing aprecise change to be introduced into the mouse genome. Also, when agene is altered, the level of functional modification is complete andunvarying from the conception of the animal until the death of thatanimal. Another advantage of this technology is that mouse linesbearing defined mutations of genes on separate chromosomes can becrossed to generate offspring with combined mutations. The knockoutapproach also has potential disadvantages. For example, the technologyseems far easier in mice than in other animals, even other rodents.Another potential disadvantage is that in models of gene inactivation,a mouse has the entire embryonic period to develop compensatorymechanisms. Thus a mouse that has never produced a protein is differentfrom an animal in which that same protein is inhibited as an adult.This problem can be partially offset through the creation ofconditional and/or tissue-specific knockouts in which gene inactivationonly occurs in the presence (or absence) of specific drugs or incertain tissues ( 31 ). Finally, knockout technology createsa permanent change in the animal's genome. This is different frompharmacological inhibitors, which can be administered in varying dosesand then withdrawn. Despite the potential limitations of the knockouttechnique, its use in creating unique genetic models has made thistechnology one of the most important in modern biological research.1 B" V* H$ u0 {
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Since 1995, targeted homologous recombination has been used to createknockout mice that are null for the individual components of the RAS( 6, 7, 17, 20, 22, 23, 25, 27, 36, 37, 43, 44, 48, 51, 53, 54, 58 ). Several recent reviews have cataloged the individual mouselines and discussed the phenotype of these animals. In this review, wewill focus on recent work from our laboratory that made use ofhomologous recombination to selectively inactivateangiotensin-converting enzyme (ACE). We develop the hypothesisthat the creation of mouse models bearing a functional RAS in only asmall subset of tissues is a powerful approach to gain insight into theorgan-specific function of the RAS.
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ACE.1: MICE NULL FOR ACE
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ACE is a carboxypeptidase that converts angiotensin I intoangiotensin II. ACE has other substrates such as bradykinin and acetyl N -acetyl-seryl-aspartyl-lysyl-proline (a putative bone marrow suppressor), but the physiological role of ACE in the regulation of these peptides is unclear. There are two ACE isozymes.Somatic ACE is made by endothelium and other somatic tissues and is the form of ACE responsible for blood pressure regulation. This protein contains two separate catalytic sites (termed NH 2 -terminaland COOH-terminal sites) that are capable of hydrolyzing angiotensin I( 3, 50, 56 ). Most somatic ACE is bound to tissuesvia a hydrophobic COOH-terminal domain ( 2 ). Endotheliumexpresses abundant ACE; thus the lung, an organ rich in endothelium,contains a high concentration of ACE. ACE protein is also produced byrenal proximal tubular epithelium, vascular adventitia, areas of the gut, activated macrophages, and selected portions of the brain ( 5, 49, 52 ). A second ACE isozyme is produced bydeveloping male germ cells ( 10 ). This protein, termedtestis ACE, is the result of a tissue-specific promoter located withinintron 12 of the ACE gene ( 21 ). Testis ACE corresponds tothe COOH-terminal half of somatic ACE and contains only theCOOH-terminal catalytic domain ( 15, 29, 30 ). As describedbelow, studies in knockout mice have proven that testis ACE plays acritical role in male fertility.

Both our group and the laboratory of Dr. Oliver Smithies created micenull for all ACE expression ( 17, 27 ). The animals made byour group are called ACE.1 (null) and produce neither somatic nortestis ACE (Fig. 1 ). ACE null mice have aprofound reduction of blood pressure. When blood pressure is assessedwith an automated tail-cuff device, a normal mouse will average105-110 mmHg. In contrast, littermate mice null for ACE have asystolic blood pressure of 73 mmHg. The magnitude of the blood pressure 30 mmHg) is striking, and, to our knowledge, mice lackingACE have a lower blood pressure than any other animal modelinvestigated to date. Maybe not surprisingly, the one exception is micelacking angiotensinogen ( 25, 43, 53 ), both AT 1 receptors (AT 1A and AT 1B ) ( 44, 54 ), or renin ( 36, 58 ), which also present with areduction in systolic blood pressure of ~30 mmHg. Despite having alifetime to regulate the many other physiological systems thatinfluence blood pressure, a mouse lacking a functional RAS presentswith a profound reduction of blood pressure. Thus no physiologicalsystem or combination of systems can compensate for the lack ofangiotensin II. The RAS is the central regulator of blood pressure, anobservation that gives insight into the remarkable clinical efficacy ofRAS inhibitors.- i' \9 t+ S7 B5 n+ ]  ~8 e  r7 w

Fig. 1. Angiotensin-converting enzyme (ACE) targeting construct. Top : wild-type organization of the ACE locus. The fourconstructs used to make the ACE knockout strains are shown next. TheACE.1 (null) and ACE.2 (tissue null) constructs are similar, becauseeach includes a neomycin cassette (Neo R ) positioned inintron 12 of the gene, along with specific mutations to disrupt testisACE expression. The ACE.2 (tissue null) construct also contains a cDNAelement encoding the COOH-terminal domain of ACE positioned 5' to theneomycin cassette. The ACE.3 (liver ACE) and ACE.4 [kidneyandrogen-regulated protein (KAP) ACE] constructs are also similar,because they were each designed to target the tissue-specificexpression of ACE. To accomplish this, a neomycin cassette was placedimmediately 5' to the transcriptional start site for somatic ACE andwas followed by a tissue-selective promoter of choice. In the case ofthe ACE.3 (liver ACE) construct, the promoter chosen was an albuminpromoter/enhancer, and, in the case of the ACE.4 (KAP ACE) construct,the chosen promoter was for the KAP. Black rectangles, exons; arrows,promoters; gray rectangles, start site and first exon of testis ACE.

In addition to low blood pressure, ACE.1 (null) mice have othercomponents to their phenotype. Perhaps most surprising was amaldevelopment of the kidney characterized by underdevelopment of therenal medulla and papilla. The origins of this lesion are not known,although elegant work by Miyazaki et al. ( 39, 40 ) suggestsimproper function of the ureter as a potential cause.* r) A. `0 w$ o. R; f( F  S

ACE.1 (null) mice also give insight into the role of testis ACE. Malemice homozygous for the ACE.1 (null) gene do not reproduce well; theyproduce few litters and each litter is far smaller than that producedby wild-type male mice. Several additional studies have definitivelyshown that it is the lack of testis ACE that is responsible for thereproductive phenotype. For example, elegant experiments by Ramaraj etal. ( 46 ) used transgenic technology to reinstate testisACE expression onto the background of ACE null mice. The restitution oftestis ACE expression restored normal fertility, even in the presenceof a reduced blood pressure due to the continued lack of somatic ACE.In contrast to male knockout mice, female ACE.1 (null) mice appear tohave relatively normal fertility.; Y- c6 y: E! X' m* r' g8 F: h7 Q

ACE.2: NO TISSUE ACE EXPRESSION: }; D0 U( U+ C$ @% ^

In creating the ACE.2 (tissue null) model, we hoped to engineer aline of mice that would express somatic ACE in the absence of testisACE (Fig. 1 ) ( 18 ). Unfortunately, our manipulation of themouse genome resulted in a stop codon being introduced after exon 12 ofthe somatic ACE gene. The resulting animal produced a truncated ACEprotein roughly one-half the size of the wild-type mouse. This proteinretained the NH 2 -terminal signal sequence (responsible forprotein export from cells) and the NH 2 -terminal catalyticregion. What this protein lacked was the COOH-terminal catalytic domainand, even more importantly, the COOH-terminal hydrophobic tail of theprotein normally responsible for anchoring the protein to cellmembranes. In summary, ACE.2 (tissue null) mice have a truncated ACEprotein that circulates in the plasma but no measurable ACE activityassociated with tissues such as the lung or the kidney. The phenotypeof ACE.2 (tissue null) mice was similar to ACE.1 (null) knockoutanimals; systolic blood pressure was in the mid-70s and the mice wereunable to concentrate urine.3 k! m; [* P8 c1 x

A comparison of ACE.1 (null) and ACE.2 (tissue null) illustratesseveral different aspects of blood pressure control. First, the ACE.2(tissue null) mice give insight into the concept of "total bodyload" of ACE. The RAS has sufficient homeostatic capacity to functionacross a wide range of ACE body load. For example, Krege et al.( 28 ) have demonstrated that mice with one, three, and evenfour copies of the ACE gene maintain normal blood pressure. However, ina normal mouse, most ACE is bound to tissues. Although the ACE.2(tissue null) mice have roughly one-third of normal levels ofcirculating plasma ACE activity, they have no tissue ACE. Thus theseanimals have a marked deficiency of total body ACE. This is reflectedin very low levels of plasma angiotensin II that are similar to thelevels seen in ACE.1 (null) mice ( 8 ). A hypothesisdeveloped from these two strains was that total quantities of body ACE(as opposed to the precise localization of ACE activity) may be thecritical factor in determining adequacy of ACE activity.
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There is another way to view the ACE.2 (tissue null) mice. Theseanimals are also deficient in specific tissue beds of ACE. Recenttheories of the RAS have proposed important physiological (andpathophysiological) roles for angiotensin II generated locally bytissues such as the vascular endothelium ( 11, 14, 41 ). ACE.2 (tissue null) mice lack endothelial ACE, and the low blood pressure in these animals may be due to a specific inability to generate vascular angiotensin II. The relative importance of these twohypotheses (total body ACE vs. precise patterns of tissue expression)is discussed below in the analysis of the ACE.3 (liver ACE) mice.

A major difference between the ACE.1 (null) and ACE.2 (tissue null)strains of mice is in renal development. As previously noted, ACE.1(null) mice have an underdevelopment of the renal medulla and papilla.In contrast, most ACE.2 (tissue null) mice do not have this defect( 18 ). Because the blood pressures of these two strains arevirtually indistinguishable, this implies that the renal malformationis attributable to other factors besides the absolute reduction ofblood pressure. Also, the difference in kidney phenotype between theACE.2 (tissue null) and ACE.1 (null) mice suggests that among theseveral different facets of the phenotype found in the ACE nullanimals, renal development is the most sensitive to amelioration by thesmall increases of ACE and angiotensin II present in ACE.2 (tissuenull) mice compared with truly null animals. The precise biochemistryof this is not at all understood.

That ACE.2 (tissue null) knockout mice have a very low blood pressurebut retain normal renal function creates a mouse model of exceptionalutility. Specifically, although ACE.1 (null) animals have abnormalmeasures of renal function, as indicated by an elevation of serumcreatinine and a reduction of creatinine clearance, ACE.2 (tissue null)mice have a normal serum creatinine and creatinine clearance( 8 ). Despite this, ACE.2 (tissue null) knockout mice arestill unable to concentrate urine. Thus in ACE.2 (tissue null) mice,the abnormal physiology must be the result of the lack of ACE andangiotensin II, as opposed to renal insufficiency. To understand themolecular basis for the inability to concentrate, we determined theabundance of several different transport proteins and ion channelstypically associated with renal concentrating ability ( 26 )(Fig. 2 ). Within the inner medulla, ACE.2(tissue null) knockout mice had a reduction of the 117-kDa ureatransporter UT-A1 (28 ± 5% of normal), the chloride channelClC-K1 (6 ± 6% of normal), and aquaporin 1 (39 ± 5% ofnormal). In contrast, the protein abundance of the water channelaquaporin-2 and the urea transporter UT-B were equivalent to levelsfound in wild-type mice. Within the outer medulla, there was a modestreduction of Na-K-Cl cotransporter isoform 1/type 1 bumetanide-sensitive Na-K-2Cl cotransporter (56 ± 11% of normal)as well as a more marked reduction of the water channel aquaporin-1(29 ± 6% of normal). Quite unexpectedly, there was also asevenfold increase in the apical potassium channel ROMK (711 ± 187% of wild type). To our knowledge, this is the first observation ofan animal with upregulation of ROMK. One explanation for this unusualfinding is as a response to the hyperkalemia that is typical in ACEknockout mice.- t' H; K; M+ {% ~6 q( n9 }
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Fig. 2. Altered medullary transport proteins in ACE.2 (tissue null) mice. Left : Western blot analysis of ClC-K1 ( A; arrow,80-kDa protein band for ClC-K1) and ROMK ( B, arrow, 45-kDaprotein band for ROMK) expression within the renal medulla of wild-type(WT) and ACE.2 (tissue null) knockout mice (KO). Right :densitometric data averaged from several mice (* P  Q  h1 m: j/ P0 }) o4 k
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In evaluating the ACE.2 (tissue null) data, the marked decrease inUT-A1 and, in particular, ClC-K1 would be consistent with an inabilityto concentrate urine. The reduction of UT-A1 may reduce the amount ofurea transported from the terminal inner medullary collecting duct.However, almost certainly, the profound reduction of ClC-K1 wouldreduce NaCl reabsorption from the thick ascending limb. Knockout micethat lack ClC-K1 (Clcnk1 mice) also present with a marked inability toconcentrate urine ( 35 ). Why mice with a substantialreduction of angiotensin II generation have a defect in the proteinconcentration of UT-A1 and ClC-K1 is not understood. However, thesedata illustrate how the creation and analysis of knockout mice havealready yielded new insights into angiotensin II-mediated regulation ofrenal function. Another new approach in the study of the concentratingdefect in ACE.2 (tissue null) mice is through micropuncture, andstudies of this type are currently under investigation.
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ANGIOTENSIN II AND ERYTHROPOIESIS
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Both ACE.1 (null) and ACE.2 (tissue null) knockout mice presentwith an ~20% reduction of both hematocrit and hemoglobin levels ( 8 ). Although one might envision that ACE.1 (null) miceare anemic partly due to renal insufficiency, ACE.2 (tissue null) miceare equally anemic despite having normal chemical measures of renalfunction. The anemia in these strains is not due to iron deficiency orapparent hemolysis. Furthermore, both ACE.1 (null) and ACE.2 (tissuenull) knockout mice have elevated serum erythropoietin levels. Theanemia in these mice is real in that a donor erythrocyte infusion using 51 Cr-labeled red blood cells demonstrated a reduction ofred cell mass associated with normal total blood volume. Perhaps evenmore informative was a study in which ACE.2 (tissue null) knockout micereceived angiotensin II during a 2-wk period by subcutaneous osmoticminipump. These mice remained deficient in ACE, despite the restorationof angiotensin II. Thus this experiment directly assessed thephysiological role of angiotensin II in the anemia, as opposed to otherpeptides that may be ACE substrates. The results were clear:restitution of angiotensin II elevated both systolic blood pressure andhematocrit to near normal levels. Although we have not eliminatedthe possibility that blood pressure itself played a role in erythrocyteformation, these studies compliment a host of clinical investigationssuggesting a direct role of the RAS in erythropoiesis ( 16, 19, 33, 55 ). It is well known that erythropoietin is dependent onthe intracellular signaling of the Jak-STAT system for efficacy.Erythropoietin knockout, erythropoietin receptor knockout, and Jak 2 knockout mice all present with the phenotype of embryonic lethality dueto severe anemia ( 24, 32, 42, 57 ). Angiotensin IIactivates Jak-STAT signaling ( 34 ). Whether this somehowprovides the link by which angiotensin II influences erythropoiesis isunder investigation.

ACE.3: SELECTIVE HEPATIC EXPRESSION OF ACE5 z# @1 y2 @6 n) D
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To summarize, the evaluation of ACE null mice revealed a complexphenotype involving changes in blood pressure, electrolyte balance,renal function, renal development, reproduction, and hematologicalparameters. Perhaps this was not surprising given the many differentorgan systems possessing receptors for angiotensin II. In fact, it isimportant to realize that an ACE null mouse is very different from awild-type animal. Put another way, the typical approach of knocking outa gene induces a major change in the physiology of that animal. Such anapproach is highly informative but not at all subtle. Indeed, the nullanimal approach has intrinsic limitations given the many differentphysiological systems that no longer operate in the knockout mouse.
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Our group has developed an approach for creating a more selectiveknockout of protein expression. The basis of the approach is asimplistic view of gene structure: the ACE gene, as with any gene, canbe modeled as two separate parts. There are the exonic portions of thegene encoding amino acid sequence and the promoter portion of the generegulating the temporal and tissue-specific expression of the protein.In principle, a modification of the promoter is capable of changing thetissue-specific patterns of gene expression. Specifically, we reasonthat if the endogenous ACE promoter is substituted with a highlytissue-specific promoter, then ACE expression would only be present inthe limited subset of tissues recognizing this new promoter. Such amouse would be null for ACE expression in other tissues not recognizingthe new tissue-specific promoter. This is similar to creating atransgenic mouse and breeding it onto a knockout background, anapproach that has been previously used to investigate thetissue-specific expression of angiotensinogen ( 13 ).Technically, we believed that our approach would be simpler for thestudy of ACE due to the reduced fertility present in the ACE knockout.Thus the goal of the ACE.3 (liver ACE) mouse was to investigate such anapproach by placing the coding portions of the ACE gene under thecontrol of the albumin promoter ( 9 ).

The ACE.3 (liver ACE) mouse was engineered to contain two DNA cassettesseparating the endogenous somatic ACE promoter from the translationstart site of the gene (Fig. 1 ). A neomycin cassette was insertedimmediately 3' of the endogenous somatic ACE promoter and acted as atranscriptional barrier to promoter activity. We inserted an albuminpromoter cassette 3' to the neomycin cassette, containing both enhancerand core promoter sequences sufficient to initiate transcription.
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Evaluation of tissue ACE activity revealed a marked difference amongwild-type, ACE.3 (liver ACE) heterozygote, and ACE.3 (liver ACE)homozygote mice (Fig. 3 ). Although awild-type mouse expresses virtually no ACE within the liver, the ACE.3(liver ACE) homozygous mouse makes abundant hepatic ACE that islocalized on the cell membranes of hepatocytes. In contrast, awild-type mouse expresses abundant ACE in the lung but the ACE.3 (liver ACE) homozygote is completely null for lung ACE activity and ACE protein. In fact, we were unable to detect any endothelial ACE expression in any tissue of the ACE.3 (liver ACE) mice. ACE.3 (liverACE) mice express no ACE in the vascular adventitia, the GI tract, thespleen, or the heart. Only in the kidney is residual ACE activityobserved, but even there, the level is only 15% that of wild-typemice. Histological examination of the kidney revealed no ACE expressionby renal endothelial cells. Rather, the residual ACE is expressed byrenal tubular epithelium. Finally, ACE.3 (liver ACE) mice express ACEin two other locations, circulating ACE in plasma presumably due toenzymatic release from hepatocytes and normal testis ACE expression,because the testis ACE promoter remained unaltered in the ACE.3 (liverACE) construction. In summary, by changing promoter control of the ACEgene, we created a mouse strain that produces no endothelial oradventitial ACE but rather makes abundant ACE in the liver, an organthat normally has none. Such an approach allowed us to directly examinethe physiological role of vascular ACE. Specifically, we asked whethersuch a mouse could adequately regulate blood pressure. What we observedwas that the ACE.3 (liver ACE) mouse was in all ways normal. Itssystolic blood pressure was indistinguishable from littermate controls. Renal development was typical. The ACE.3 (liver ACE) concentrated urineequivalently to a wild-type mouse. Serum electrolyte values wereunremarkable, and the animal had both a normal hematocrit and a normalreproductive function. Indeed, evaluation of angiotensin II peptidelevels within the plasma shows that the ACE.3 (liver ACE) mouse hadequivalent levels to those of a wild-type mouse.

Fig. 3. Tissue-specific ACE expression in the ACE.3 (liver ACE) strain.ACE.3 (liver ACE) wild-type ( / ), heterozygote ( / ), and knockout( / ) mice were killed, and ACE activity was measured in organhomogenates. Total organ ACE activity was calculated for the liver,lungs, and kidneys of each mouse and normalized for the mass of themouse. The livers of ACE.3 (liver ACE) / mice have abundant totalACE activity, due in part to the large size of this organ. In contrast,ACE.3 (liver ACE) / mice have no detectable ACE activity in lung.ACE.3 (liver ACE) / mice have ~14% of the renal ACE found inwild-type mice. Values are group means ± SE.

What are the implications of this analysis? First, vascular ACEexpression is not obligatory for normal blood pressure control. Indeed,with sufficient ACE in the liver, the intrinsic regulatory capacity ofthe kidney can maintain homeostasis. Such a result again emphasizes theconcept of total ACE body load. We hypothesize that sufficient ACE inany location within the body, coupled with the normal renal homeostaticmechanisms, is sufficient to result in normal blood pressure control.Second, ACE.3 (liver ACE) mice produced a concentrated urine despitehaving only 15% of normal renal ACE. Although we cannot conclude thatrenal ACE has no intrinsic function, it seems clear that the kidney canoperate normally with even small amounts of renal ACE. Finally, theACE.3 (liver ACE) mouse is valuable in establishing the validity ofpromoter substitution in creating strains of mice with very selectedexpression of ACE.
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The ACE.3 (liver ACE) mouse also showed some of the potential drawbacksof the promoter substitution approach. First, the residual ACEexpression in the kidney was probably due to the intrinsic propertiesof the albumin promoter used in the construct. Mice transgenic forsimilar promoter constructs also showed slight renal expression of areporter protein ( 45 ). Also, the principal site of ACEexpression in the ACE.3 (liver ACE) mouse was liver hepatocytes, anorgan and tissue that does not normally produce the protein. It isconceivable that such ectopic expression of ACE could influence thephenotype of the animal. Another drawback of the ACE.3 (liver ACE) micewas substantial plasma ACE, which was 80% of normal. We believe thisprotein originates in a fashion similar to the naturally circulatingACE present in wild-type mice, namely, as a result of proteolyticcleavage of ACE from the surface of cells ( 2 ). However, wewould like to find a means of reducing both the circulating ACE levelsand the breakthrough expression of ACE in the kidney. One possibilityis to create a compound heterozygous animal in which one ACE allelewill be from the ACE.1 (null) mouse while the second ACE allele will be ACE.3 (liver ACE). In theory, such a compound heterozygous animal should produce liver ACE with reduced ACE activity in both the plasmaand the kidney.6 y9 t/ x( T) p! q

ACE.4: KIDNEY ANDROGEN-REGULATED PROTEIN PROMOTER DRIVING ACE
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A strategic goal is to create an animal with ACE expressionlimited to the kidney. Such an animal would be informative about specific renal functions of the RAS vs. systemic effects. One animalcreated for this purpose is termed ACE.4 [kidney androgen-regulated protein (KAP) ACE] and has the KAP promoter-driving somatic ACE expression (Fig. 1 ). Normally, KAP is highly responsive to androgen andis found in proximal tubular epithelium and the uterus ( 38, 47 ). Although the KAP promoter was satisfactory in sometransgenic experiments ( 12 ), it proved inadequate totarget ACE expression in the ACE.4 (KAP ACE) mouse. Specifically, inthe absence of exogenous androgens, virtually no ACE expression wasmeasured in the tissues of either male or female ACE.4 (KAP ACE) mice. With exogenous androgens, renal ACE expression remained normalvalues. Thus ACE.4 (KAP ACE) mice had a profound reduction of somaticACE and presented with a phenotype nearly identical to that of theACE.1 (null) mouse. For example, the blood pressure of seven ACE.4 (KAPACE) homozygotes averaged 70.0 ± 3.3 mmHg, a value very similarto that observed in the ACE.1 (null) and ACE.2 (tissue null) knockouts.The essential characteristics of the ACE.1-4 strains aresummarized in Table 1. The results with ACE.4 (KAP ACE) mice indicate that the promoter substitution approach is highly dependent on the characteristics of individual promoter constructs. As opposed to transgenic mice, only a single copy of thetissue-specific promoter is inserted immediately upstream of the ACEgene. In the case of the KAP promoter, this proved unable to drive ACEexpression.

Table 1. Phenotypic characteristics of ACE knockout mice
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SUMMARY. l& s9 S/ W) n

Targeted homologous recombination has the power to create newstrains of mice expressing the RAS in selected subsets of tissues. Notonly will these new genetic models be useful for the study of bloodpressure regulation, but they also have great promise in theinvestigation of the function of the RAS in complicated disease models.For example, the ACE.3 (liver ACE) mouse has a normal blood pressure inthe absence of endothelial or adventitial expression of ACE. One canenvision experiments in which this animal is used to derive insightinto the function of the RAS in disease processes such asatherosclerosis or diabetes. By choosing which tissues of the mousewill express a functional RAS, we can perform subtle yet elegantexperiments to evaluate individual functions of a system that has manydifferent physiological roles. Such an approach may reveal yetadditional subtle actions of the RAS in the control of both normal andaberrant physiology.
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ACKNOWLEDGEMENTS

The authors thank Dr. Mario Capecchi (University of Utah) and Dr.Pierre Corvol (College de France) for help with these studies.
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