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Departments of *Internal Medicine, 
Neuroscience and Cell Biology, and Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas.
Correspondence to Dr. Peter Igarashi, Division of Nephrology, UT Southwestern, 5323 Harry Hines Blvd., MC8856, Dallas, TX 75390-8856. Phone: 214-648-2754; Fax: 214-648-2071; E-mail: peter.igarashi{at}utsouthwestern.edu
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Abstract |
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-galactosidase exclusively in the kidney, although the expression pattern is incomplete (Am J Physiol 277: F599–F610, 1999). To further define the region that mediates tissue-specific expression, transgenic mice carrying 1341 bp or 324 bp of the 5' flanking region linked to a green fluorescent protein (GFP) reporter gene were produced. Transgenic mice carrying 1341 bp of the 5' flanking region expressed GFP in all embryonic tissues that endogenously express Ksp-cadherin, including the ureteric bud, Wolffian duct, Müllerian duct, and developing tubules in the mesonephros and metanephros. In the adult kidney, GFP was highly expressed in thick ascending limbs of Henle’s loops and collecting ducts and weakly expressed in proximal tubules and Bowman’s capsules. Transgenic mice carrying 324 bp of the 5' flanking region exhibited expression exclusively in tubular epithelial cells in the developing kidney and GU tract. Immunoblot analysis showed that the expression of GFP was restricted to the kidney in adult mice. Taken together, these results demonstrate that 324 bp of the Ksp-cadherin 5' flanking region is sufficient to direct epithelial-specific expression in the developing kidney and GU tract. Transgenic mice that express GFP in the mesonephros, metanephros, ureteric bud, and sex ducts may be useful for cell lineage studies. |
Introduction |
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The expression of Ksp-cadherin in the kidney (metanephros) is developmentally regulated. Ksp-cadherin mRNA is first abundantly expressed in the mouse metanephros at E14.5 (Vanden Heuvel G and Igarashi P, unpublished observations). Expression increases during late gestation and remains high in the adult kidney. The protein is first expressed in developing nephrons after the S-shaped body stage (5). There is no expression in earlier nephric structures, including pretubular aggregates, renal vesicles, or comma-shaped bodies. Ksp-cadherin is expressed in the tubular portion of developing nephrons but is absent from the glomerular anlage. Ksp-cadherin is also expressed in the ureteric bud, which is an epithelial outgrowth from the mesonephric duct that is the anlage of the renal collecting system. Within the branching ureteric bud, the expression in maturing collecting ducts is higher than in the immature ampullae. Thus, the expression of Ksp-cadherin is differentiation-dependent as well as tissue-specific.
In addition to the metanephros, Ksp-cadherin is transiently expressed during embryogenesis in tubular epithelial cells of the developing GU tract. Ksp-cadherin transcripts have been detected by in situ hybridization in epithelial cells of the mesonephros, renal pelvis, and ureter (6). Transcripts are also expressed in the Wolffian (mesonephric) duct, which gives rise to the epididymis and vas deferens in males, and the Müllerian (paramesonephric) duct, which gives rise to the oviducts, uterus, cervix, and vagina in females. A low level of mRNA expression has also been reported in the developing lung (6), but expression of Ksp-cadherin protein has not been detected in this tissue by antibody staining (R. Thomson, Yale Medical School, personal communication). The expression of Ksp-cadherin in the developing GU tract is transient, because there is no expression in the adult renal pelvis, ureter, or male and female genital tracts.
Although the biologic function of Ksp-cadherin remains unknown, it has proven to be a useful model of kidney-specific gene expression. To begin understanding the basis for its tissue-specific and developmentally regulated expression, we have previously cloned and characterized the promoter of the mouse Ksp-cadherin gene (7). The promoter is "TATA-less," but it contains multiple GC-boxes and a CAAT box. The promoter also contains consensus recognition sites for several tissue-specific transcription factors, including basic-helix-loop-helix proteins, GATA factors, HNF-1, HNF-3, and C/EBP. Reporter gene assays in transiently transfected cells have shown that the Ksp-cadherin promoter can direct expression of a luciferase reporter gene in two renal epithelial cell lines (MDCK, mIMCD-3) but not in two mesenchymal cell lines (NIH3T3, MMR1), which indicates that the activity of the promoter is cell-specific (7). Transgenic mice carrying 3425 bp of the Ksp-cadherin 5' flanking region linked to a lacZ reporter gene express -galactosidase exclusively in the kidney, verifying that the promoter is tissue-specific in vivo (8). However, the expression of the lacZ reporter gene is restricted to renal collecting ducts, whereas Ksp-cadherin is endogenously expressed in all nephron segments. In transgenic embryos, lacZ is expressed in the branching ureteric bud but is not expressed in developing nephrons. Taken together, these results demonstrate that 3425 bp of the Ksp-cadherin 5' flanking region directs tissue-specific expression in vitro and in vivo. However, this fragment of the promoter produces an incomplete expression pattern.
The minimal Ksp-cadherin promoter that is required for promoter activity in transfected renal epithelial cells has been defined by deletion analysis (7). MDCK cells and mIMCD-3 cells were transiently transfected with various lengths of Ksp-cadherin 5' flanking region linked to a luciferase reporter gene. Truncation of the promoter from -2680 bp to -250 bp did not significantly affect luciferase activity. However, further deletion to -113 bp reduced luciferase activity by 40%, and truncation to -31 bp completely abolished activity. These results suggest that the proximal 250 bp of the promoter is sufficient for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays have shown that this region of the promoter contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells (7). Promoter mapping in transfected cultured cells may produce different results from expression in transgenic mice, and only studies in animals can determine the developmental timing and cell-specificity of expression within an organ and in all tissues; the present study was therefore undertaken to narrow down the region of the Ksp-cadherin 5' flanking region that mediates tissue-specific and developmentally regulated expression in vivo. We find that 1341 bp of the 5' flanking region recapitulates the complete expression pattern of the Ksp-cadherin gene. Transgenes containing 324 bp of the 5' flanking region exhibit variegating position effects but are also expressed exclusively in kidney tubules and the developing GU tract.
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Materials and Methods |
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Plasmid Construction
A reporter plasmid (BgEGFP) containing a human -globin minimal promoter (-37 bp to +12 bp), GFP reporter gene, and bovine growth hormone polyadenylation signal was produced by digesting the plasmid BGZA (9) with NcoI and XhoI and replacing the 3.5-kb restriction fragment containing the lacZ gene and SV40 polyadenylation signal with the 1.1-kb NcoI/XhoI restriction fragment from pIRES-EGFP (Clontech, Palo Alto, CA). A GFP reporter plasmid (pKsp1.3/BgEGFP) containing 1341 bp of the Ksp-cadherin 5' flanking region (nucleotides 2430–3770 of GenBank accession no. AF118228) was produced by digesting the plasmid pKsp(1268F)-luc (7) with HindIII, isolating the 1.3-kb restriction fragment, end-filling with Klenow, and cloning the fragment into a unique SmaI site upstream to the -globin minimal promoter in BgEGFP. A GFP reporter plasmid (pKsp0.3/BgEGFP) containing 324 bp of the Ksp-cadherin 5' flanking region (nucleotides 3447–3770) was produced by digesting the plasmid pKsp(250F)-luc (7) with HindIII, end-filling with Klenow, and then digesting with NheI. The 0.3-kb restriction fragment was isolated and cloned into unique XbaI and SmaI sites upstream to the -globin minimal promoter in BgEGFP. The sequences of the plasmid inserts were verified by DNA sequencing. The plasmids were tested by transient transfection into mIMCD-3 renal epithelial cells. Fluorescence microscopy revealed expression of GFP in transfected cells but not in untransfected cells (data not shown).
Generation of Transgenic Mice
Transgene fragments were isolated from vector sequences by digestion with NotI and XhoI followed by agarose gel electrophoresis, electroelution, and purification by anion-exchange chromatography (Elutip-d; Schleicher & Schuell, Keene, NH). The purified DNA was concentrated in Microcon-30 filters (Millipore, Bedford, MA), resuspended at a concentration of 2 to 3 µg/ml in microinjection buffer (10 mM Tris-Cl [pH 7.40], 0.25 mM EDTA), and sterilized by filtration through 0.2-µm filters. Transgene DNA was microinjected into the pronuclei of fertilized oocytes as described previously (8,9). Fertilized oocytes were from B6D2F1 crosses or ICR donors. Some microinjections were performed by the UT Southwestern Transgenic Mouse Core Facility. Microinjected embryos were transferred into the oviducts of pseudopregnant foster mothers and were permitted to develop to term. Transgenic progeny were identified by PCR analysis of tail DNA. The primers used for amplification of the GFP gene were (5'-ACAAGTTCAGCGTGTCCGGC and 5'-TTGTGGCTGTTGTAGTTGTACTCCAG). PCR conditions are available upon request. Southern blot analysis was performed to verify transgene integration and the absence of rearrangements of the transgene. The expression of the transgene was analyzed in F1 progeny derived from crosses with ICR mice or B6D2 hybrids. Embryos were obtained from time-pregnant mice in which the morning of detection of the copulatory plug was designated as E0.5, and gestational age was verified using Theiler’s criteria (10). All experiments involving animals were performed in accordance with APS guidelines and under the auspices of the Institutional Animal Care & Research Advisory Committee.
Green Fluorescent Protein
The expression of GFP was examined by fluorescence microscopy of fresh, unfixed tissues and embryos. Tissues were sectioned at 50 to 100 µm using a Vibratome 1500 system (St. Louis, MO) and were visualized under epifluorescence illumination using a Nikon TE300 inverted microscope equipped with a standard FITC filter set (excitation, 460 to 500 nm; dichroic mirror, 505 nm; emission, 510 to 560 nm). Embryos were obtained by Caesarean section from timed-pregnant mice at E9.5 to E17.5 and were hand-dissected to expose the kidney and GU tract before examination. Thin sections were prepared from tissues and embryos by perfusion-fixation and/or immersion for 1 h in phosphate-buffered saline (PBS) containing 4% paraformaldehyde. Specimens were cryoprotected overnight in 30% sucrose, embedded in OCT, frozen in isopentane, and sectioned at 5 to 10 µm using a Leica Frigocut 2800N cryostat. Sections were mounted with aqueous medium (Vectashield) and visualized using a Nikon TE300 or Zeiss Axioplan 2 microscope. Photomicrographs were obtained using Spot or Zeiss digital cameras and a Power Macintosh computer equipped with Adobe Photoshop and Openlab software. As negative controls, samples from nontransgenic littermates were photographed under identical exposure conditions.
Immunostaining and Lectin Staining
Immunostaining and indirect immunofluorescence were performed as described previously (8). Five- to ten-micrometer-thick cryosections were prepared from formaldehyde-fixed tissues, mounted, and incubated with primary antibody for 1 h at room temperature. An affinity-purified rabbit antibody to green fluorescent protein (generous gift from Dr. Pam Silver, Harvard Medical School, Cambridge, MA) was used at a dilution of 1:2000 (11). A rabbit antibody to mouse Tamm-Horsfall protein (generous gift from Dr. John Hoyer, University of Pennsylvania, Philadelphia, PA) was used at a dilution of 1:2000 (12). Sections were then washed with PBS and incubated with a 1:200 dilution of Alexa Fluor 488–coupled or Alexa Fluor 594–coupled goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Stained sections were mounted with Vectashield and examined using a Nikon TE300 or Zeiss Axioplan 2 microscope.
Lectin staining was performed on 10-µm-thick cryosections of kidneys and embryos as described previously (8). Collecting ducts and ureteric buds were labeled by staining with TRITC-conjugated Dolichos biflorus agglutinin (DBA). Proximal tubules were labeled by staining with TRITC-coupled Lotus tetragonolobus agglutinin (LTA). After washing with PBS containing 0.1% bovine serum albumin, the sections were postfixed in PBS containing 4% paraformaldehyde, rinsed in PBS, and then mounted with Vectashield. Photomicrographs were obtained as described above. Images of double-labeled sections were merged using Openlab or Adobe Photoshop software.
In Situ Hybridization
Radioactive in situ hybridization was performed on paraffin-embedded sections using 35S-radiolabeled RNA probes as described previously (13). Antisense and sense riboprobes were transcribed from a 900-bp mouse Ksp-cadherin cDNA cloned in the plasmid pCR2.1. After hybridization and washing, the slides were dipped in emulsion, exposed, and developed in Kodak D19. Photomicrographs were taken with a Leica M420 macroscope equipped with a darkfield condenser.
Organ Culture
Metanephric organ culture was performed using an adaptation of a previously described method (14). Metanephroi were isolated from embryos at E12.5 and were placed on transparent Cyclopore filters. Metanephroi were cultured for up to 7 d at the air-medium interface in DMEM/Ham’s F12 containing 10% fetal calf serum. Cultures were photographed under phase-contrast and epifluorescence illumination using a Nikon TE300 inverted microscope.
Immunoblot Analyses
Tissue samples were homogenized for 20 s in 5 volumes (wt/vol) of PBS containing protease inhibitors (Complete Mini Protease Inhibitor Cocktail; Roche). Samples were centrifuged (10 min; 16000 x g), and the supernatant was frozen for 2 h at -20°C. After thawing, centrifugation was repeated. Protein concentration was determined using the Coomassie protein reagent (Pierce, Rockford, IL) with bovine serum albumin as the standard. Protein samples (100 µg) were reduced (100 mM DTT; 70°C; 10 min), analyzed by SDS-PAGE using 4 to 12% Bis-Tris gels (Invitrogen, Carlsbad, CA), and electrophoretically transferred to nitrocellulose membranes (Hybond-C, Amersham, Gaithersburg, MD). Membranes were blocked in PBS containing 5% nonfat dry milk and 0.05% Tween 20, and they were then incubated in the same solution containing rabbit anti-GFP antibody (1:1000), monoclonal mouse anti Ksp-cadherin antibody (1:1000; generous gift from Dr. Brent Thomson, Yale Medical School, New Haven, CT), or rabbit anti LRP-85 antibody (1:1000; generous gift from Dr. Joachim Herz, University of Texas Southwestern, Dallas, Texas) (15). Bound antibodies were detected with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:2000) and enhanced chemiluminescence (SuperSignal, Pierce).
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Results |
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-globin minimal promoter and a green fluorescent protein (GFP) reporter gene were analyzed in transgenic mice. Figure 1 shows the structures of the transgenes. The -globin minimal promoter is transcriptionally inactive in transgenic mice in the absence of added enhancers (16). Therefore, if the test fragments contain tissue-specific regulatory elements that can activate the heterologous -globin promoter, tissue-specific expression of the GFP reporter gene will be observed. The test fragments also contain the Ksp-cadherin transcription start site; therefore, activation of the homologous promoter will also be detected in this assay. GFP, an intrinsically fluorescent protein from A. victoria, was selected as the reporter because it can be visualized in living cells without the addition of exogenous substrates and does not interfere with embryonic development. For these studies, we used a variant of GFP (EGFP) with a red-shifted excitation spectrum and humanized codon usage to permit detection with a standard fluorescein filter set and to increase expression in mammalian cells (17). Transgenic mice were generated by pronuclear microinjection and were bred to create permanent lines. As shown in Table 1, multiple independent founders were obtained for each transgene. With a few exceptions (noted below), the multiple founders for each transgene exhibited identical patterns of expression.
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Examination of thin cryosections of a transgenic kidney under fluorescence microscopy confirmed that GFP was expressed in tubular epithelial cells. In the outer medulla, GFP was highly expressed in thick ascending limbs of Henle’s loops and medullary collecting ducts but was absent from blood vessels within the vascular bundles (Figure 2G). In the renal cortex, the overall level of expression was lower than in the medulla (Figure 2H). GFP was expressed at high levels in cortical thick ascending limbs of Henle’s loops and cortical collecting ducts and was weakly expressed in proximal tubules and the parietal epithelia of Bowman’s capsules. No fluorescence signal was detected in glomerular tufts or in the kidney from a nontransgenic littermate (Figure 2I). Four independent founders that expressed the Ksp1.3/BgEGFP transgene were produced. Three founders expressed GFP in the pattern described above, and one exhibited partial expression in the renal medulla.
Expression of the Ksp1.3/BgEGFP Transgene in Renal Tubules
To verify that the green fluorescence signal was due to the expression of GFP, thin sections of the kidneys from Ksp1.3/BgEGFP hemizygous mice were immunostained with an antibody directed against the protein (11). Figure 3A shows a section of the renal cortex from a transgenic mouse stained with a GFP primary antibody and an Alexa Fluor 488–coupled secondary antibody. Only tubular epithelial cells stained positive with the antibody. There was no staining of glomerular tufts, blood vessels, or renal interstitial cells. The kidney from a nontransgenic littermate showed no significant staining under identical conditions (Figure 3D), indicating that the antibody staining was specific. To identify the GFP-positive tubules in panel A, the section was costained with TRITC-coupled Lotus tetragonolobus (LTA), a lectin that specifically labels proximal tubules (18). The merged image (Figure 3C) shows expression of GFP in LTA-positive proximal tubules. Proximal tubules in the renal cortex expressed moderate levels of GFP, whereas proximal tubules in the outer medulla, which probably represent S3 segments, showed minimal expression (Figure 2G).
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Expression of the Ksp1.3/BgEGFP Transgene in Mouse Embryos
The expression of Ksp-cadherin in the kidney and GU tract is developmentally regulated. To test whether the expression of the Ksp1.3/BgEGFP transgene was regulated similarly, transgenic embryos were examined at different gestational ages. Figure 4A shows fluorescence microscopy of a transgenic embryo at E15.5, a stage at which Ksp-cadherin is expressed in the kidney (metanephros), ureter, and sex ducts. Green fluorescence signal was observed in the metanephros, ureter, and Wolffian duct but not in the testis or surrounding tissues. No significant fluorescence was observed in the kidney from a nontransgenic littermate (Figure 4B). To verify the expression of GFP, transgenic embryos were sectioned and stained with a specific antibody. As shown in Figure 4C, immunostaining was restricted to the developing kidney (metanephros) and GU tract. There was no expression of GFP in any of the surrounding tissues, including the liver, intestine, and rectum, which verified that the expression of the transgene was tissue-specific.
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The Ksp1.3/BgEGFP transgene was also expressed in the developing GU tract. Figure 4I shows the expression of GFP in tubular epithelial cells of the developing ureter, Wolffian duct, and Müllerian duct. Expression in these embryonic structures was transient, however, because there was no expression in the adult renal pelvis, ureter, or male and female genital tracts (data not shown). Taken together, these results demonstrate that the developmental profile of the Ksp1.3/BgEGFP transgene was identical to the endogenous expression of Ksp-cadherin.
Expression of the Ksp0.3/BgEGFP Transgene in Embryos and Newborn Mice
Next, we expressed transgenes containing a 324-bp fragment of the 5' flanking region of the Ksp-cadherin gene. This region contains the 250-bp promoter that had previously been shown to be sufficient for promoter activity in transiently transfected renal epithelial cells (7). Transgenic mice carrying the 324-bp fragment linked to a GFP reporter gene were produced by pronuclear microinjection, and the expression of GFP in the hemizygous progeny was examined by fluorescence microscopy and immunostaining. Figures 5A and 5B show fluorescence microscopy of the kidneys from newborn mice derived from two independent founders. Both mice exhibited GFP fluorescence in the kidney, whereas no fluorescence was detectable in the kidneys from nontransgenic littermates (not shown). Moreover, no fluorescence was observed in the heart from a transgenic mouse (Figure 5C), indicating that the expression of GFP was kidney-specific. A higher magnification image of the renal medulla from a transgenic mouse shows that GFP was expressed in collecting ducts (Figure 5D). Figures 5E and 5F show the expression of GFP in a transgenic embryo at E17.5. GFP was expressed only in the metanephros and not in the stomach, pancreas, intestine, and adrenal gland, which verified that the expression was tissue-specific in the developing embryo. Within the metanephros, the expression of GFP was restricted to tubular epithelial cells. However, in contrast to transgenes containing the 1341-bp fragment of the 5' flanking region, which were expressed uniformly in renal tubules (except for one founder that exhibited partial expression), all the founders carrying the 324-bp fragment exhibited variegated expression in which the transgene was expressed in some, but not all, the cells. Figures 5G through 5I show that GFP was expressed in a variegated pattern in developing loops of Henle, collecting ducts, and proximal tubules. In addition to variegated expression, one founder exhibited ectopic expression in the developing lung and skin (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to expression in the adult kidney, the Ksp1.3/BgEGFP transgene is also expressed in the developing kidney and GU tract. The transgene is expressed in tubular epithelial cells of the mesonephros, metanephros, ureteric bud, and sex ducts. Expression in the developing ureter and sex ducts is transient, because there was no expression in the adult ureter or genital tracts. In the developing metanephros, the transgene is expressed in the branching ureteric bud and in developing nephrons after the S-shaped body stage. There is no expression in early nephric structures, including condensed mesenchyme, renal vesicles, and comma-shaped bodies. The expression in the developing metanephros, mesonephros, and sex ducts is identical to the expression of the Ksp-cadherin mRNA and protein reported previously (5,6). Therefore, the 1341-bp fragment of 5' flanking region contains regulatory elements that are sufficient to completely recapitulate the tissue-specific and developmentally regulated expression of the Ksp-cadherin gene.
Although other tissue-specific promoters have been shown to direct transgene expression in the ureteric bud and renal collecting system (e.g., Pax-2, aquaporin-2, Hoxb7), the 5' flanking region of the Ksp-cadherin gene is the first genomic region that has been shown to direct tissue-specific expression in developing renal tubules in the mesonephros and metanephros. The 5' flanking region should be useful for expressing any gene of interest in kidney tubules in transgenic mice. One example of this type of experiment is described in a companion article (20), in which the 1341-bp fragment was used to express Cre recombinase in the developing kidney and GU tract. Interestingly, the 1341-bp fragment of the Ksp-cadherin 5' flanking region produces a more complete expression pattern than the longer 3425-bp fragment described previously (8). Transgenes containing the 3425-bp fragment are expressed in a tissue-specific pattern, but the expression is incomplete and confined to collecting ducts and ureteric bud. The 3425-bp fragment may contain cis-acting element(s) that inhibit transgene expression in mesonephric tubules and developing nephrons.
Ksp0.3/BgEGFP transgenic mice carrying a 324-bp fragment of the 5' flanking region also express GFP only in the developing kidney and GU tract. However, caution is necessary in the interpretation of the results with the 324-bp promoter. In contrast to the uniform expression produced by the 1341-bp fragment, the 324-bp fragment produces a variegated expression pattern. Variegated expression is frequently observed in transgenic mice and reflects transgene silencing in a fraction of the cells. The mechanism is poorly understood, but it is believed to involve epigenetic modification of the transgene due to chromosomal position effects (21). The 324-bp region may lack an insulator element or locus control region that militates against variegating position effects. Despite the presence of variegating position effects, our studies show that transgenes containing 324 bp of the Ksp-cadherin 5' flanking region are expressed exclusively in the kidney and GU tract in developing embryos. Immunoblot analysis verifies that the expression of the transgene is kidney-specific in adult mice. Taken together, these results demonstrate that a 324-bp fragment of Ksp-cadherin 5' flanking region linked to a -globin minimal promoter and GFP reporter gene exhibits tissue-specific expression in transgenic mice.
The 324-bp fragment is the shortest genomic fragment that has been demonstrated to have kidney-specific activity in transgenic mice. Because of the variegated expression pattern, further deletion analysis of the region was not attempted. Future experiments will be directed at identifying the transcription factors that bind to the 324-bp fragment and that mediate tissue-specific gene expression. The 324-bp region contains a (CA)n microsatellite repeat that is not conserved in the human promoter and is therefore unlikely to be essential for tissue-specific expression. A 120-bp sequence that is >80% identical between mouse and human is more likely to contain essential elements. Recent studies have shown that this region contains a functional binding site for hepatocyte nuclear factor 1 (HNF-1), a homeodomain protein that is involved in kidney-specific gene expression (22).
Although GFP has been used as a reporter gene in transgenic mice, there are relatively few descriptions of its use in the kidney. Transgenes containing GFP under the regulation of the renin promoter are expressed in juxtaglomerular cells and several extrarenal tissues (23). Transgenes containing the Hoxb7 promoter and GFP reporter gene are expressed in the ureteric bud but also in the brain and snout (24). CX/GFP transgenes containing a cytomegalovirus enhancer and -actin/-globin promoter are expressed in podocytes as well as skeletal muscle, pancreas, and heart (25). Transgenes containing the THP promoter linked to a GFP reporter gene exhibit kidney-specific expression in thick ascending limbs of Henle’s loops and early distal tubules (26). The current study further supports the utility of GFP as a reporter gene in the kidney. Expression can be detected by fluorescence microscopy of unfixed tissues, cultured organs, or formaldehyde-fixed tissues as well as by immunostaining. Because the expression of GFP can be detected in living cells, Ksp1.3/BgEGFP transgenic mice may be useful for future cell lineage studies. For example, pluripotent stem cells isolated from Ksp1.3/BgEGFP transgenic mice could be induced to differentiate, and the expression of GFP could be monitored as an indicator of tubular epithelial differentiation.
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Acknowledgments |
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(HNF-1). Am J Physiol Renal Physiolpublished May, 7, 2002, 10.1152/ajprenal.00128.2002
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