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【摘要】 Here we describe the generation and characterization of a mouse strain that expresses an improved Cre (iCre) recombinase ( 48 ) under the control of the endogenous 11 -hydroxysteroid dehydrogenase type 2 (11HSD2) promoter. Progeny of 11HSD2/iCre and ROSA26 reporter mice were used to determine the pattern of iCre expression by measuring the activity of the LacZ gene product -galactosidase in a panel of tissues. On Cre recombinase activity, intense -galactosidase activity (X-gal staining) was observed in the classic mineralocorticoid target segments of the kidney, as well as in the colon, and both female and male reproductive organs. Weaker iCre expression was detected in the lung and heart. In the brain, strong iCre activity was present in cardiovascular centers that are known to express 11HSD2 and mineralocorticoid receptors [nucleus tractus solitarius (NTS) and amygdala] as well as in the granular layer of the cerebellum. iCre expression was weaker in neonatal kidney and colon than in the adult but was present in the hair follicles and cartilage. These results indicate that in the 11HSD2/iCre strain iCre expression faithfully represents the expression pattern of endogenous 11HSD2. Thus this mouse model represents the first Cre deleter strain that can be used to eliminate desired genes in every mineralocorticoid target tissue. This mouse model should serve as a useful resource for investigators who want to study the function of genes involved in aldosterone action and genes in other pathways that are selectively expressed in these cells.
【关键词】 aldosterone lox P collecting duct kidney
ONE OF THE MOST POWERFUL APPROACHES to assess the pathophysiological role of a gene is to examine the consequences of its disruption using genetically modified animals. However, global disruption of a gene often results in embryonic lethality or a complex phenotype from which the tissue-specific functions of the targeted gene cannot be easily evaluated. The best strategy to overcome these difficulties is to generate spatially restricted somatic mutations using the Cre/ lox P ( 29, 44 ) or FLP/FRT ( 40 ) systems.
Spatially regulated Cre expression seems particularly important in the case of very heterogeneous tissues such as the kidney, which consists of several nephron segments of different embryonic origins, and even within segments there is cellular heterogeneity. Some renal gene products are expressed in a restricted fashion (e.g., the thiazide-sensitive Na/Cl cotransporter is exclusively expressed in the distal convoluted tubule; Refs. 1, 34 ), while some other genes, although not expressed in the entire nephron segment, are still crossing segmental boundaries, such as the epithelial sodium channel (ENaC), which is expressed in the connecting tubule and the collecting duct system ( 8 ). To study the in vivo roles of such gene products necessitates a panel of mouse strains in which Cre is expressed in different nephron segments or cell types. However, the existing mouse models expressing Cre in renal cells ( 32, 45, 52, 56 ) do not provide a full coverage. Thus to determine the physiological functions of the ever increasing number of genes found to be differentially regulated within the kidney, a larger arsenal of tissue-, segment-, or cell-specific Cre-expressing strains is needed.
Aldosterone, the main mineralocorticoid hormone, is a key regulator of Na balance. The effects of aldosterone are mediated via the mineralocorticoid receptor (MR), a ligand-dependent transcriptional factor that is expressed in the renal connecting segment and collecting ducts ( 26 ). However, defining the roles of MR-regulated genes is hindered by an additional problem, i.e., that the MR is also expressed in tissues that are not classic aldosterone targets ( 9, 21 ). Interestingly, because the MR has the same high affinity for aldosterone as for cortisol (and corticosterone), whether or not an MR-expressing cell responds to aldosterone depends on the presence of the enzyme 11 -hydroxysteroid dehydrogenase (11HSD2; Refs. 10, 43 ). 11HSD2 inactivates intracellular glucocorticoids, thereby allowing aldosterone to occupy the MR. 11HSD2 is expressed at high levels in aldosterone target cells, e.g., principal cells of the collecting duct express 2-3 million 11HSD2 molecules ( 31 ) (vs. several thousand MRs).
On the other hand, some extrarenal MR-expressing tissues (e.g., the hippocampus or the heart) do not seem to express 11HSD2 ( 9 ); thus such tissues are not aldosterone sensitive. The importance of extrarenal MRs has been underscored by recent clinical trials such as the RALES and EPHESUS studies in which blockade of MRs resulted in a dramatic improvement of patients with chronic heart failure ( 35, 36 ). This clinical significance makes studying the function of MR-regulated genes in vivo even more relevant. However, this complex question necessitates models in which the function of MR-regulated genes can be selectively eliminated in aldosterone-sensitive vs. aldosterone-insensitive tissues.
Here we describe the generation and characterization of a mouse strain that expresses an improved Cre (iCre) recombinase ( 48 ) under the control of the endogenous 11HSD2 promoter, and therefore iCre expression faithfully represents the expression pattern of 11HSD2.
MATERIALS AND METHODS
Generation of the Targeting Construct
Figure 1 summarizes the 11HSD2/iCre targeting construct. A plasmid encompassing 12,191 bp of the mouse 11HSD2 locus that includes the entire exon 1 and part of exon 2 was generated from two overlapping genomic clones, generously provided by Dr. Yuri Kotelevtsev (University of Edinburgh, Edinburgh, Scotland, UK; Ref. 19 ). This plasmid was mutagenized to convert the initiator ATG to a Sal I site and to eliminate the Age I site residing in intron 1. The region between the new Sal I site and the remaining Age I site in exon 1 was then replaced by a 2.8-kb Xho I- Age I DNA fragment carrying the iCre codons (a generous gift of Dr. R. Sprengel, Max-Planck Institute for Medical Research, Heidelberg, Germany; Ref. 48 ) and a neomycin resistance (neoR) expression cassette bracketed by two unidirectional FRT sites. To select against random genomic integration, the final targeting construct was generated by adding expression cassettes for diphtheria toxin A (DTA) and thymidine kinase (TK) from herpes simplex to the 5'- and 3'-ends of the genomic sequence, respectively.
Fig. 1. Knockin strategy to target improved Cre (iCre) into the 11 -hydroxysteroid dehydrogenase-2 (11HSD2) locus. The targeting construct is shown at top, and the structure of the mouse 11HSD2 gene with exons in thick lines and nos. is shown at bottom. The lengths of the left and right homology arms are indicated with dashed lines above the construct. The position of the DNA sequence used as external probe is marked as 3'P. The dashed lines below correspond to the Xho I and Hind III wildtype fragments hybridizing with 3'P. The size of these fragments in the mutant allele is shown in parenthesis.
Generation of 11HSD2/iCre Knockin Mice
All experimental protocols were approved by the Dartmouth Institutional Animal Care and Use Committee, and all procedures using experimental animals were carried out adhering to the American Physiological Society's Guiding Principles in the Care and Use of Animals. Knockin mice were generated by the Transgenic and Knockout Mouse Core Facility at Dartmouth. After linearization with Not I, the targeting construct was electroporated into R1129/Sv x 129/Sv-CP embryonic stem (ES) cells. Recombinant clones were enriched by positive selection with G418 and by negative selection with DTA and gancyclovir.
Recombinant clones were then identified by Southern blotting using DNA digested with Hind III or Xho I and an external probe corresponding to the distal part of exon 2, intron 2, and the proximal portion of exon 3. Clones with dual hybridization signals at 3.8 and 6.3 kb and 8.1 and 5.9 kb, respectively, were further characterized by long-range PCR and then microinjected into C57bl6-derived blastocysts and implanted into pseudopregnant foster females. Progeny were identified by PCR analysis of tail DNA (PCR conditions are available on request), and Southern blot analysis was used to verify integration of the transgene.
Following germline transmission of the modified allele, the neomycin expression cassette was removed in vivo by crossing the 11HSD2/Cre mice with FLPe mice ( 40 ).
Determination of -Galactosidase Activity
To determine the pattern of iCre activity, HSD2/iCre mice were crossed with the reporter strain B6;129-Gtrosa26 tmSor (ROSA26; Jackson Laboratories, ME), and tail DNA from pups was analyzed by PCR to identify bitransgenic animals (expressing both a single copy of the knockin iCre gene and the ROSA26 reporter). Tissues from bitransgenic and control mice were fixed in 4% paraformaldehyde on ice for 2 h and then cryoprotected overnight in 30% sucrose and embedded in O.C.T., and 10-µm cryosections were made. -Galactosidase activity was detected by staining with 2 mg/ml 5-bromo-4-chloro-3-indoyl- - D -galactoside (X-gal) at 37°C for 2 h, followed by mounting with Permount mounting medium.
Immunohistochemistry
Indirect immunofluorescence was performed on kidney cryosections using kidney segment-specific markers, as follows. To identify collecting ducts, we used a rabbit polyclonal antibody against aquaporin-2, and to identify distal convoluted tubules, we used an antibody against the thiazide-sensitive Na/Cl cotransporter (these 2 antibodies were a generous gift of Dr. Mark Knepper, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD, and have been previously characterized; Refs. 16, 33 ). A guinea pig antibody against the Na/Ca exchanger (generously supplied by Dr. R. Reilly; Yale University School of Medicine, New Haven, CT) was used as a marker of the connecting tubules ( 38 ). Endogenous 11HSD2 expression was identified using a rabbit antibody against 11HSD2, generously supplied by Dr. Brian Reeves (Pennsylvania State College of Medicine, Hershey, PA; Ref. 22 ). Sections were first stained for X-gal and then rinsed five times in PBS, overlayed with the primary antibodies, and incubated at room temperature for 1 h. Sections were rinsed again with PBS and incubated with AlexaFluor568- or FITC-labeled secondary antibody for 1 h. The stained sections were mounted in Vectashield and inspected by bright-field and fluorescence microscopy. Images were captured on a PXL cooled CCD camera (Photometrics) attached to an Olympus IMT2 microscope equipped with an epifluorescence attachment and standard FITC, 4',6-diamidino-2-phenylindole (DAPI), and Texas red filter sets, using a x 60 planapo objective (N.A. 1.4, Nikon).
RESULTS
Analysis of Cre Expression and Activity in 11HSD2/iCre Knockin Mice
Cre expression in aldosterone target cells. Immunohistochemistry of Cre expression alone does not provide information on the function of Cre, i.e., its ability to excise a floxed gene. To evaluate the ability of 11HSD2/iCre mice to mediate tissue-specific Cre/lox recombination, these mice were bred to the Cre reporter strain ROSA26, which carries the lacZ reporter gene that is activated on Cre/lox recombination ( 51 ). Thus tissues with Cre activity will express -galactosidase activity (i.e., X-gal staining).
Since in the mouse strain we generated, iCre transcription is driven by the endogenous 11HSD2 promoter, we expected to see Cre-mediated recombination in aldosterone target tissues where 11HSD2 protects the MR from occupation by glucocorticoids ( 10, 43 ). The main target organ of aldosterone is the kidney. Indeed, as shown in Fig. 2, the kidneys of the progeny of 11HSD2/iCre and ROSA26 mice exhibited abundant X-gal staining in a minority tubule population in the cortex and medulla. The location of these tubules suggested that they correspond to connecting and collecting tubules, which are the main mineralocorticoid target segments. The majority of tubules in the cortex, corresponding to proximal tubules, were negative. Similarly, there was no staining in glomeruli or connecting tissue. Sections from mice carrying only the ROSA26 reporter gene were negative for X-gal staining (not shown).
Fig. 2. Cre-mediated recombination in the kidney of double-transgenic HSD2/iCre/ROSA26 reporter mice. X-gal staining of adult mouse shows intense blue staining in a minority population of tubules in the cortex and medulla.
To verify that the expression pattern of the Cre activity corresponds to endogenous 11HSD2 expression, we performed immunohistochemistry, costaining kidney sections with X-gal and an antibody against 11HSD2 ( 22 ). Higher magnification images shown in Fig. 3, A and B, demonstrate perfect colocalization of the two stains, indicating that the Cre-expressing nephron segments also express 11HSD2. Further identification of the X-gal-positive tubules was obtained by costaining sections for aquaporin-2, which is expressed only in the collecting ducts and connecting tubules ( 17, 24 ). Figure 3, C and D, shows colocalization of aquaporin-2 and X-gal staining in these nephron segments. It should be noted that Cre expression (i.e., X-gal staining) is more widespread than that of aquaporin-2, in accordance with previous findings on the broader renal distribution of 11HSD2 than aquaporin-2 ( 22, 30 ). As expected, cells of the connecting tubule were also strongly positive for X-gal. Figure 3, E and F, demonstrates X-gal staining in tubules that reacted with an antibody against Na/Ca exchanger (a connecting tubule marker; Ref. 38 ). 11HSD2 activity was shown to be present in distal convoluted tubules in the rabbit ( 53 ). However, costaining of kidney sections with an antibody against the thiazide-sensitive Na/Cl cotransporter (TSC1) that is solely expressed in the distal convoluted tubule ( 1, 34 ) and X-gal illustrates the lack of colocalization of these markers ( Fig. 3, G and H ). Our results are in agreement with previous findings by Campean at al. ( 4 ) demonstrating no endogenous 11HSD2 expression in the mouse distal convoluted tubule.
Fig. 3. Cre-mediated recombination in the kidney is restricted to the aldosterone-responsive distal nephron in double-transgenic HSD2/iCre/ROSA26 mice. Kidney sections were costained with X-gal ( A, C, E, G ) and an antibody against 11HSD2 ( B ), aquaporin-2 ( D ), the connecting tubule marker Na/Ca exchanger ( F ), or the thiazide-sensitive Na/Cl cotransporter TSC1 ( H ). A, C, E, and G are light microscopic images of the same areas of kidney sections shown in B, D, F, and H, respectively. 11HSD2-driven iCre activity showed perfect colocalization with endogenous 11HSD2 immunoreactivity ( A, B ) and was localized to the connecting and collecting duct system ( A-F ) but not to the distal convoluted tubule ( G, H ).
In addition to the kidney, 11HSD2 is expressed in other sodium-transporting epithelia such as the colon, small intestine, and salivary glands ( 13 ). As shown in Fig. 4 A, iCre excision was detected in the colon (both in the epithelial and in the external muscular layers). The jejunum and salivary glands were also positive for X-gal staining (not shown). In addition, our results show that iCre excision did occur in the lung ( Fig. 4 B ), a tissue in which low levels of both 11HSD2 and MR expression were detected previously in the human ( 13 ).
Fig. 4. Cre-mediated recombination in nonrenal tissues expressing mineralocorticoid receptor (MR). X-gal staining was observed in colon ( A ) and in certain cells of the lung ( B ) as well as in the heart ( C ). T, trachea; L, lung.
iCre expression in tissues that are not classic aldosterone targets. MRs are expressed in the heart, but the presence of 11HSD2 in cardiac cells is currently debated ( 9, 46 ). Interestingly, as shown in Fig. 4 C, cardiomyocytes of the progeny of 11HSD2/iCre x ROSA26 mice in both the atria and ventriculum exhibited medium-to-weak X-gal staining, indicating low yet detectable levels of 11HSD2 promoter activity in the mouse heart. It should be noted, however, that the level of expression was much lower than in the kidney, and X-gal staining was not detected in all cells ( Fig. 4 C ).
In addition to classic aldosterone target cells, 11HSD2 is also expressed in organs that lack MRs, such as the uterus, the ovary ( 3, 41 ), and the epididymal epithelium in the mouse ( 54 ). Accordingly, our results demonstrate significant iCre expression in these tissues. In the epididymis, X-gal staining was strongest in the caput region ( Fig. 5 A ), but weaker staining could be detected in the entire epididymis. In the uterus, where 11HSD2 presumably protects the developing embryo from high maternal glucocorticoids ( 3, 41 ), strong X-gal staining was detected in the myometrium and somewhat weaker staining in the endometrium ( Fig. 5 B ). iCre activity was also present in the ovaries (data not shown). 11HSD2 is expressed in the adrenal glands in the rat but not in the mouse ( 6 ), and we also failed to detect iCre activity in the mouse adrenals (not shown).
Fig. 5. Cre-mediated recombination in reproductive organs and the brain. Strong blue staining was observed in the caput epididymis and weaker staining in other regions of the epididymis ( A ), in the uterus ( B ) and in the external granular layer of the cerebellum ( C ). Note that X-gal staining was also observed in other regions of the brain, listed in Table 1.
Tissues that are known to lack 11HSD2 expression, including the skeletal muscle, liver, spleen, ureter, urethra, and urinary bladder ( 28 ), had no detectable iCre activity.
iCre expression in the brain. Previous work clearly demonstrated that most MR-expressing cells in the brain, such as the hippocampus, lack 11HSD2 ( 39 ). However, a few MR-expressing nuclei, including the nucleus tractus solitarius (NTS), amygdala, subcommissural organ, the ventromedial nucleus of the hypothalamus, and locus coeruleus, have been reported to express 11HSD2 ( 39 ), suggesting that, in these sites, MR is activated by aldosterone. 11HSD2 expression in the brain is broader in the embryo than in the adult ( 39 ), reflecting the importance of protecting the developing central nervous system from high concentrations of glucocorticoids. Our data on the localization and intensity of iCre expression in the brain of the progeny of HSD2/iCre x ROSA26 mice are summarized in Table 1. X-gal staining was intense in the NTS and amygdala, corresponding to the previously described expression of 11HSD2 ( 39 ). In the medulla, punctated X-gal staining was also observed in the external cuneate nuclei. In the cerebellum, strong X-gal staining was detected in the external granular layer ( Fig. 5 C ), in agreement with a recent report showing high 11HSD2 expression in these cells in newborn mice ( 39 ). In the pons/midbrain region, high activity was observed in the pontine reticular formation as well as in pontine nuclei and periaqueductal gray, while in the diencephalons/forebrain region, in addition to amygdala, iCre activity was present in the hypothalamic region and in several thalamic nuclei. In agreement with the previously reported absence of 11HSD2 expression in the hippocampus and the cerebral cortex ( 39 ), these regions were also negative for X-gal staining.
Table 1. Locations of -galactosidase activity in the brain of 11HSD2/iCre x ROSA26 mice
iCre expression in neonatal mice. It is generally accepted that the expression pattern of 11HSD2 is dynamic during development and is more widespread in the embryo than in the adult ( 2 ). Interestingly, the two organs that have perhaps the highest 11HSD2 activity in the adult, the kidney and the colon, were found to lack 11HSD2 expression in newborn mice ( 7 ). Similarly, we found very low iCre expression in the kidneys and colon of 1- to 3-day-old pups (data not shown). On the other hand, we found high iCre expression in the cerebellum, in the hair follicles ( Fig. 6 A ), and in the cartilage in P1-3 mice ( Fig. 6 B ). MR expression has not been shown for the cartilage in adult human, mouse, or rat, but both MR and 11HSD2 expression has been described for developing hair follicles in humans ( 12 ) and in the cerebellum of mice ( 15 ).
Fig. 6. Cre-mediated recombination in neonatal tissues. A : hair follicle. B : cartilage.
DISCUSSION
We describe here a novel mouse strain that expresses an iCre recombinase under the control of the endogenous promoter of the 11HSD2 enzyme. Although several Cre-expressing strains have been generated that can be used to inactivate genes carrying loxP recognition sequence in certain segments of the kidney, such as the kidney specific-cadherin, aquaporin-2, Hoxb7, or sglt-2 promoter-driven transgenic Cre strains ( 32, 42, 45, 56 ), the HSD2/iCre strain is the first one in which inactivation of "floxed" genes expressed in every mineralocorticoid target cell will be possible. Thus these animals should serve as a useful resource for investigators studying the function of aldosterone-regulated genes. This strain should be particularly well suited to inactivate genes that are expressed not only in classic aldosterone targets but also in other tissues, and their elimination in the nonaldosterone target sites results in embryonic or early postnatal lethality.
In contrast to traditional methods that use mice overexpressing Cre transgenes, we knocked in Cre recombinase in the endogenous 11HSD2 locus. Transgenes driven by "tissue-specific" promoters often exhibit a pattern of expression that is different from that directed by the endogenous promoter, because of differences in landing site or the number of transgenes. To obtain animals that express Cre only in the desired location, typically, many different transgenic lines need to be tested and characterized. Furthermore, expression of transgenes is often variegated, even if they are expressed in the desired tissue. By use of the "knockin" strategy, these problems can be overcome, since the regulatory elements driving tissue-specific expression are maintained in their proper chromosomal contexts. Indeed, expression of iCre in the strain we generated seems to faithfully represent the expression pattern of the endogenous 11HSD2 gene. A potential disadvantage of the knockin strategy is that the targeted gene can be disabled. However, this disadvantage becomes less of a concern if one allele is sufficient for normal function. This is the case with 11HSD2, as animals with only one 11HSD2 allele have normal phenotype ( 19 ). In addition, with judicious choice of the targeted gene, this "disadvantage" can be turned into an advantage, if it facilitates the study of the phenotype of the gene to be knocked out. For instance, the function of genes that regulate epithelial Na + reabsorption and blood pressure, such as ENaC, serum- and glucocorticoid-induced kinase 1, etc., would be expected to become "constitutively" active (i.e., over-active) in mice in which endogenous 11HSD2 activity is decreased, and thus the MRs become activated by glucocorticoids.
An additional concern is the possibility of interference from elements residing in the selection cassette, which can also affect the pattern of expression or function of the targeted or neighboring genes. To minimize such an effect, in the present study, we bracketed the neo resistance cassette by FRT sites, which allowed the excision of this region in vivo by crossing the 11HSD2/Cre mice with FLPe mice ( 40 ).
In this study, we used an iCre recombinase generated by applying the mammalian codon usage to Cre recombinase, which greatly enhanced its expression levels ( 48 ). This is an important improvement, since a critical determinant of the success of generating tissue-specific knockout animals is the level of expression of Cre recombinase in the desired tissue. The iCre gene also circumvents another problem, i.e., the high CpG content of the prokaryotic coding sequence, which often resulted in epigenetic silencing in mammalian cells. iCre recombinase has been used in mice to generate several types of tissue-specific knockouts ( 5, 23, 48 ).
The expression pattern of 11HSD2 protein has been well characterized in human and rat using immunohistochemistry ( 3, 13, 14, 20, 22 ). There is much less information on 11HSD2 expression in mice, and most of the available information is at the level of RNA expression ( 6, 28 ). In the present study, we provide a comprehensive analysis of the expression pattern of 11HSD2 in adult and neonatal mice, using the activity of the endogenous 11HSD2 promoter as a read-out. Our results indicate that the 11HSD2 promoter-driven expression of iCre corresponds well with the previously reported 11HSD2 expression pattern in aldosterone target cells and in the brain. Importantly, our study also revealed novel sites of 11HSD2 expression in mice. For instance, we observed strong iCre expression in the neonatal cartilage and in the external muscle layer of the intestine. The presence of 11HSD2 in these tissues has been previously unknown. Because in our studies, iCre driven by 11HSD2 is already active during the embryonic life, these findings might reflect 11HSD2 expression during development in precursor cells that are not present in the adult. It also should be noted that X-gal staining can represent 11HSD2 expression at any time during development, since the 11HSD2-driven iCre activity results in permanent excision of the stop codon from the -galactosidase gene present in the ROSA26 reporter mouse ( 51 ).
The presence of 11HSD2 in the heart is currently controversial. There are reports describing 11HSD2 activity in human cardiac tissue ( 25 ) as well as in the heart of rats ( 18, 27, 49 ), while others failed to show the presence of either mRNA or protein in the rat or mouse heart ( 28, 47 ). Our data demonstrate 11HSD2-driven iCre expression in mouse cardiomyocytes. The fact that little or no expression was observed in neonatal hearts, and, even in adults, excision occurred in only a fraction of the cells, suggests that 11HSD2 expression is relatively modest. Nevertheless, this 11HSD2 activity could be functionally relevant, since it appears that cortisol- and aldosterone-bound MRs in cardiomyocytes mediate different, even opposing, effects ( 37 ). Thus, although the moderate cardiac 11HSD2 expression may not be sufficient to give the heart full protection from endogenous glucocorticoids, nevertheless it could facilitate aldosterone occupancy of the MR at a more favorable aldosterone-to-cortisol ratio, for instance, during severe volume depletion.
Our study found no 11HSD2-driven Cre activity in the pancreas, where previously 11HSD2 expression was reported in the human and rat ( 13, 50 ). The reason for this apparent discrepancy is not clear, but apart from species differences, it might be due to the different methodologies in those studies vs. the present work. For instance, expression of LacZ in the pancreas might not have been enough to detect -galactosidase activity after Cre recombination. Alternatively, the immunohistochemical methods in previous studies might have used antibodies that cross-reacted with other antigens.
In the brain, we observed 11HSD2-driven Cre activity in regions that were previously reported to have 11HSD2 activity, such as the NTS, amygdala, and certain thalamic nuclei ( 39 ). These areas also express MR; thus 11HSD2 ensures aldosterone selectivity to mediate central actions on blood pressure and salt appetite. In addition, our results demonstrate high Cre activity in the granular layer of the cerebellum. MRs are expressed mostly in the Purkinje cells ( 11 ) but not in the external granular layer. Nevertheless, because aldosterone synthase is also expressed in the cerebellum ( 55 ), it is possible that 11HSD2 ensures aldosterone (perhaps produced locally) access to those MRs. Alternatively, 11HSD2 might protect the cells of the cerebellum from the detrimental effects of high glucocorticoids ( 39 ). A recent report showed that the cerebellar size was smaller in HSD2 null mice, because of decreases in size of both the molecular and internal granular layers ( 15 ).
An interesting question is the role of HSD2 in tissues that do not express MR, such as the reproductive tract. Our results show 11HSD2-driven Cre activity in both female and male reproductive tissues in accordance with previous reports demonstrating 11HSD2 expression or activity ( 3, 41, 54 ). We speculate that 11HSD2 expression in these organs provides a mechanism to maintain specificity of androgen- or progesterone-regulated gene expression. This is necessary, since the glucocorticoid, progesterone, and androgen receptors all bind to the same hormone response elements, and thus activation of the glucocorticoid receptor could lead to inappropriate activation of genes normally regulated by sex steroids.
In summary, we developed a novel mouse strain that expresses an iCre in known mineralocorticoid tissues and particular regions of the brain. Thus this strain should serve as a useful tool for investigators examining the consequences of eliminating genes specifically in those organs and cells ( 16, 33 ).
GRANTS
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41841 (A. Náray-Fejes-Tóth), DK-58898 and DK-55845 (G. Fejes-Tóth).
ACKNOWLEDGMENTS
We thank Dr. Mary Niblock for help with brain histochemistry, Dr. Steve Fiering for helpful discussions, Donna Morneau for excellent technical assistance, Drs. Yuri Kotelevtsev and R. Sprengel for generously providing plasmids, and Drs. Brian Reeves, Mark Knepper, and Robert Reilly for providing antibodies.
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作者单位：Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire