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Murine Cytomegalovirus Immediate-Early Promoter Directs Astrocyte-Specific Expression in Transgenic Mice

1999; Elsevier BV; Volume: 154; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)65320-5

1525-2191

Sonomi Aiba-Masago, Satoshi Baba, Ren-Yong Li, Yuichiro Shinmura, Isao Kosugi, Yoshifumi Arai, Masahiko Nishimura, Yoshihiro Tsutsui,

Toxoplasma gondii Research Studies

Murine cytomegalovirus (MCMV), which causes acute, latent, and persistent infection of the natural host, is used as an animal model of human cytomegalovirus (HCMV) infection. Transcription of MCMV immediate-early (IE) genes is required for expression of the early and late genes and is dependent on host cell transcription factors. Cell-type-specific expression activity of the MCMV IE promoter was analyzed in transgenic mice generated with the major IE (MIE) enhancer/promoter involving nucleotides −1343 to −6 (1338 bp) connected to the reporter gene lacZ. Distinct expression was observed in the brain, kidneys, stomach, and skeletal muscles. Weak expression was observed in a portion of the parenchymal cells of the salivary glands and pancreas, and expression was hardly detected in the lungs, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, and bone marrow. The spectrum of organs positive for expression was narrower than that of the HCMV MIE promoter-lacZ transgenic mice reported previously and showed a greater degree of cell-type specificity. Interestingly, astrocyte-specific expression of the transgene was observed in the brain and primary glial cultures from the transgenic mice by combination of β-galactosidase (β-Gal) expression and immunostaining for cell markers. However, the transgene was not expressed in neurons, oligodendroglia, microglia, or endothelial cells. Furthermore, the β-Gal expression in glial cultures was stimulated significantly by MCMV infection or by addition of calcium ionophore. These observations indicated that expression activity of the MCMV IE promoter is strictly cell-type specific, especially astrocyte-specific in the brain. This specific pattern of activity is similar to that of natural HCMV infection in humans. Murine cytomegalovirus (MCMV), which causes acute, latent, and persistent infection of the natural host, is used as an animal model of human cytomegalovirus (HCMV) infection. Transcription of MCMV immediate-early (IE) genes is required for expression of the early and late genes and is dependent on host cell transcription factors. Cell-type-specific expression activity of the MCMV IE promoter was analyzed in transgenic mice generated with the major IE (MIE) enhancer/promoter involving nucleotides −1343 to −6 (1338 bp) connected to the reporter gene lacZ. Distinct expression was observed in the brain, kidneys, stomach, and skeletal muscles. Weak expression was observed in a portion of the parenchymal cells of the salivary glands and pancreas, and expression was hardly detected in the lungs, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, and bone marrow. The spectrum of organs positive for expression was narrower than that of the HCMV MIE promoter-lacZ transgenic mice reported previously and showed a greater degree of cell-type specificity. Interestingly, astrocyte-specific expression of the transgene was observed in the brain and primary glial cultures from the transgenic mice by combination of β-galactosidase (β-Gal) expression and immunostaining for cell markers. However, the transgene was not expressed in neurons, oligodendroglia, microglia, or endothelial cells. Furthermore, the β-Gal expression in glial cultures was stimulated significantly by MCMV infection or by addition of calcium ionophore. These observations indicated that expression activity of the MCMV IE promoter is strictly cell-type specific, especially astrocyte-specific in the brain. This specific pattern of activity is similar to that of natural HCMV infection in humans. Human cytomegalovirus (HCMV), a member of the herpes virus group, is the most significant infectious cause of congenital anomalies of the central nervous system (CNS) caused by intrauterine infection in humans.1Weller TH The cytomegalovirus: ubiquitous agents with protean clinical manifestation.N Engl J Med. 1971; 285: 203-214Crossref PubMed Scopus (385) Google Scholar, 2Becroft DMO Prenatal cytomegalovirus infection: epidemiology, pathology, pathogenesis. Perspectives in Pediatric Pathology.in: Rosenberg HS Bernstein J Infectious Diseases. Vol. 6. Masson Publishing USA, New York1981: 203-241Google Scholar, 3Bale JF Human cytomegalovirus infection and disorders of the nervous system.Arch Neurol. 1984; 41: 310-320Crossref PubMed Scopus (126) Google Scholar, 4Ho M Congenital and perinatal human cytomegalovirus infections.in: Cytomegalovirus: Biology and Infection. ed 2. Plenum Press, New York1991: 205-227Crossref Google Scholar In adults, infection with HCMV is usually asymptomatic in immunocompetent hosts, but the virus can cause severe or fatal diseases in immunocompromised patients.5Britt WJ Alford CA Cytomegalovirus.in: Fiels BN Knipe DM Howley PM Virology. ed 3. Lippincott-Raven, Philadelphia1996: 2493-2523Google Scholar Cytomegalovirus (CMV) demonstrates a strict host cell type and species specificity in terms of infection.6Griffith PD Grundy JE Molecular biology and immunology of cytomegalovirus.Biochem J. 1987; 241: 313-324PubMed Google Scholar, 7Mocarski Jr, ES Cytomegalovirus and their replication.in: Fiels BN Knipe DM Howley PM Virology. ed 3. Lippincott-Raven, Philadelphia1996: 2447-2492Google Scholar Due to the limitations of studying HCMV in an animal host, murine CMV (MCMV) has been used as a model of human infection for pathogenesis, tissue tropism, and latency studies in the laboratory mouse.8Mocarski Jr, ES Kemble GW Recombinant cytomegaloviruses for study of replication and pathogenesis.Intervirology. 1996; 39: 320-330PubMed Google Scholar, 9Staczek J Animal cytomegaloviruses.Microbiol Rev. 1990; 54: 247-265PubMed Google Scholar, 10Tsutsui Y Kashiwai A Kawamura N Kadota C Microphthalmia and cerebral atrophy induced in mouse embryos by infection with murine cytomegalovirus in midgestation.Am J Pathol. 1993; 143: 804-812PubMed Google Scholar, 11Tsutsui Y Developmental disorders of the mouse brain induced by murine cytomegalovirus: animal models for congenital cytomegalovirus infection.Pathol Int. 1995; 45: 91-102Crossref PubMed Scopus (48) Google Scholar Similar to HCMV and other herpesviruses, MCMV genes are expressed in three sequential phases: immediate-early (IE), early, and late.6Griffith PD Grundy JE Molecular biology and immunology of cytomegalovirus.Biochem J. 1987; 241: 313-324PubMed Google Scholar, 7Mocarski Jr, ES Cytomegalovirus and their replication.in: Fiels BN Knipe DM Howley PM Virology. ed 3. Lippincott-Raven, Philadelphia1996: 2447-2492Google Scholar The expression of IE genes is dependent on appropriate cellular transcription factors that bind to the DNA sequence of the CMV major IE enhancer/promoter.12Nelson JA Gnann JW Wahren B Regulation and tissue-specific expression of human cytomegalovirus.Curr Top Microbiol Immunol. 1990; 154: 75-100Crossref PubMed Scopus (78) Google Scholar, 13Ghazal P Nelson JA Transcription factors and viral regulatory proteins as potential mediators of human cytomegalovirus pathogenesis.in: Becker Y Darai G Huang E-S Molecular Aspects of Human Cytomegalovirus Diseases. Springer-Verlag, Berlin1993: 360-383Crossref Google Scholar However, by DNA transfection, the major IE (MIE) promoter activates heterogeneous genes at high levels in most cultured cell lines. Two groups, Koedood et al14Koedood M Fichtel A Meier P Mitchell PJ Human cytomegalovirus (HCMV) immediate-early enhancer/promoter specificity during embryogenesis defines target tissues of congenital HCMV infection.J Virol. 1995; 69: 2194-2207Crossref PubMed Google Scholar, 15Fritschy J-M Brandner S Aguzzi A Koedood M Lüscher B Mitchell PJ Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice.J Neurosci. 1996; 16: 2275-2282Crossref PubMed Google Scholar and Baskar et al,16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google Scholar, 17Baskar JF Smith PP Ciment GS Hoffmann S Tucker C Tenney DJ Colberg-Poley AM Nelson JA Ghazal P Developmental analysis of the cytomegalovirus enhancer in transgenic animals.J Virol. 1996; 70: 3215-3226Crossref PubMed Google Scholar reported that expression of HCMV MIE promoter-lacZ genes in transgenic mice was cell type specific during embryogenesis. In adult tissues, the transgenes were expressed in neurons, choroid plexus cells, and endothelial cells but not in astrocytes unless the cells were stimulated.15Fritschy J-M Brandner S Aguzzi A Koedood M Lüscher B Mitchell PJ Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice.J Neurosci. 1996; 16: 2275-2282Crossref PubMed Google Scholar In the present study, we examined cell-type-specific activity of the MCMV MIE enhancer/promoter (hereafter referred as MCMV MIE promoter) in adult transgenic mice. As MCMV can infect mice as a host, transcriptional activity of the MCMV MIE promoter in transgenic mice seems to be more natural in terms of using host cellular factors than that of the HCMV MIE promoter. We used a longer enhancer/promoter sequence (nucleotides −1343 to −6; 1338 bp) of the MCMV MIE gene rather than the HCMV promoter for transgenic mice reported previously.14Koedood M Fichtel A Meier P Mitchell PJ Human cytomegalovirus (HCMV) immediate-early enhancer/promoter specificity during embryogenesis defines target tissues of congenital HCMV infection.J Virol. 1995; 69: 2194-2207Crossref PubMed Google Scholar, 15Fritschy J-M Brandner S Aguzzi A Koedood M Lüscher B Mitchell PJ Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice.J Neurosci. 1996; 16: 2275-2282Crossref PubMed Google Scholar, 16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google Scholar, 17Baskar JF Smith PP Ciment GS Hoffmann S Tucker C Tenney DJ Colberg-Poley AM Nelson JA Ghazal P Developmental analysis of the cytomegalovirus enhancer in transgenic animals.J Virol. 1996; 70: 3215-3226Crossref PubMed Google Scholar, 18Schmid EV Christoph G Zeller R Leder P The cytomegalovirus enhancer: a pan-active control element in transgenic mice.Mol Cell Biol. 1990; 10: 4406-4411PubMed Google Scholar We showed that expression activity of the MCMV IE promoter was more strictly cell type specific than that of the HCMV MIE promoter in transgenic mice, and the expression was astrocyte specific but not neuron specific. The coding region for abundant IE transcription of MCMV was reported to be restricted to a region between map units 0.769 and 0.817 of the genome.19Marks JR Mercer JA Spector DH Transcription of mouse embryo cells permissively infected by murine cytomegalovirus.Virology. 1983; 131: 247-254Crossref PubMed Scopus (15) Google Scholar, 20Keil GM Ebeling-Keil A Koszinowski UH Temporal regulation of murine cytomegalovirus transcription and mapping of viral RNA synthesized at immediate early times after infection.J Virol. 1984; 50: 784-795PubMed Google Scholar MCMV (Smith strain) DNA21Ebihara K Minamishima Y Protective effects of biological response modifiers on murine cytomegalovirus infection.J Virol. 1984; 51: 117-122PubMed Google Scholar was digested with BamHI and cloned into pACYC184. The plasmid clone containing the major IE enhancer/promoter of ie1 and ie3 (MCMV-MIE pro1) was selected using PCR with primers specific for the ie1 gene. The fragment was further digested with PstI, and the 2.3-kb fragment was subcloned into pUC18. The MCMV-MIE pro1 was excised as a KpnI-HpaI fragment covering nucleotides −1343 to −6 (1338 bp) and then inserted into the pnlacF vector containing the nuclear localization signal from SV40, Escherichia coli β-galactosidase (β-Gal) coding sequence, and poly(A) and intron from mouse protamine, provided by Dr. R. D. Palmiter, University of Washington.22Mercer EH Hoyle GW Kapur RJ Brinster RL Palmiter LD The dopamine β-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.Neuron. 1991; 7: 703-716Abstract Full Text PDF PubMed Scopus (252) Google Scholar The resulting plasmid was designated as pMCMV-MIEpro1-lacZ. The transgene fragment (5.1 kb) for microinjection was isolated from the digested plasmid DNA with KpnI and BglII. Transgenic mice were produced by standard techniques.23Hogan B Costantini F Lacy E Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986: 153-186Google Scholar Purified linearized DNA of the MCMV-MIEpro1-lacZ DNA was injected into the pronuclei of fertilized ova derived from BDF1 (C57BL/6 female × DBA/2 male) mice. After injection, ova were transferred to oviducts of pseudopregnant females of ICR mice. Transgenic founders were identified by PCR of genomic DNA prepared from tail tips using standard procedures.24Innis MA Gelfand DH Sninsky JJ White TJ PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990Google Scholar PCR was performed with MCMV-MIEpro1-lacZ-specific primers (5′ primer, 5′-GGCACGCATTCTATTGGCT-3′, and 3′primer, 5′-TTTGAGGGGACGACGACAG-3′; Figure 1, A and B) and with the mouse apolipoprotein (apo)E internal control primers (5′ primer, 5′-AACCGCTTCTGGGATTACCT-3′; 3′ primer, 5′-CATAGTGTCCTCCATCAGTG-3′) for 30 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 74°C. The reaction products were run on a 2.0% agarose gel that was then stained with ethidium bromide and photographed. Transgenic founders identified by PCR were confirmed by Southern blotting. The genomic DNA and control DNA containing nontransgenic mouse tail DNA with 1 or 10 genomic equivalents of MCMV-MIEpro1-lacZ were digested with BamHI, which cuts twice in the transgene, run on 0.9% agarose gels, and transferred on nylon membranes (MSI, Westborough, MA). The filters were then probed with 33P-labeled transgene BamHI fragment and analyzed with a Bio Imaging Analyzer (BAS1000Mac, Fuji Photo Film). Founder lines were established from transgenic mice that transmitted the MCMV-MIEpro1-lacZ to progeny. Expression analysis was performed on F1 mice obtained by mating hemizygous transgenic animals with outbred C57BL/6Cr. For histochemical analysis, adult mice (1 to 2 months old) were anesthetized with diethyl ether and perfused with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) in 0.1 mol/L phosphate buffer (PB). After perfusion, tissues were removed and fixed in freshly prepared 4% PFA and 0.1% GA in PB for 1 hour at 4°C with shaking. The tissues were incubated in a reaction mixture containing 1 mg of 5-bromo-4-chloro-3-indolyl-β-galactosidase (X-Gal) per ml, 5 mmol/L K4Fe(CN)6, 5 mmol/L K3Fe(CN)6 in PB, pH 7.3, overnight at room temperature.22Mercer EH Hoyle GW Kapur RJ Brinster RL Palmiter LD The dopamine β-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.Neuron. 1991; 7: 703-716Abstract Full Text PDF PubMed Scopus (252) Google Scholar After staining, each tissue was embedded in paraffin and sectioned. Sections were lightly stained with hematoxylin. For immunohistochemical analysis, adult mice were anesthetized and perfused with 4% PFA in 0.1 mol/L PB. Tissues were removed, fixed with 4% PFA in 0.1 mol/L PB again for 1 to 2 hours, embedded in paraffin, and sectioned. After deparaffinization and rehydration, sections were pretreated with 0.3% hydrogen peroxidase and incubated with goat serum blocking solution for 10 minutes. The sections were incubated with rabbit anti-β-galactosidase antibody (anti-β-Gal; Cappel, Durham, NC) for 30 minutes at room temperature, sequentially incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and then colored with 3-amino-9-ethyl carbazole (AEC; DAKO, Tokyo, Japan).25Shinmura Y Aiba-Masago S Kosugi I Li R-Y Baba S Tsutsui Y Differential expression of the immediate-early and early antigens in neuronal and glial cells of developing mouse brains infected with a murine cytomegalovirus.Am J Pathol. 1997; 151: 1331-1340PubMed Google Scholar Primary cortical glial cell cultures were prepared according to the methods of Togashi et al26Togashi H Sasaki M Frohman E Taira E Ratan RR Dawson TM Dawson VL Neuronal (type I) nitric oxide synthase regulates nuclear factor κB activity and immunologic (type II) nitric oxide synthase expression.Proc Natl Acad Sci. 1997; 94: 2676-2680Crossref PubMed Scopus (198) Google Scholar and Norris et al27Norris JG Tang L Sparacio SM Benveniste EN Signal transduction pathways mediating astrocyte IL-6 induction by IL-1β and tumor necrosis factor-α.J Immunol. 1994; 152: 841-850PubMed Google Scholar with some modifications. Briefly, brains were removed from postnatal day 1 transgenic mice. The cortex was dissected out under a microscope and dissociated in plating medium (Eagle's minimal essential medium (MEM); Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 10% horse serum, 20 mmol/L glucose, 2 mmol/L glutamine, 30 mmol/L NaHCO3, 100 U/ml penicillin, and 50 μg/ml streptomycin). After digestion with 0.125% trypsin (Gibco BRL) in calcium-, magnesium-free phosphate buffered-saline (PBS) and 30 mmol/L glucose at 30°C for 30 minutes, the cells were suspended in the plating medium and plated on coverslips (18 × 12 mm) in 12-multiwell plates (Becton-Dickinson, Franklin Lakes, NJ), precoated with poly-d-lysine (0.1 mg/ml; Sigma), and placed in a 5% CO2, humidified 37°C incubator. The cultures were allowed to grow to confluence, and the cells were maintained in growth medium that was similar to the plating medium but lacked FCS. For stimulation of the transgene in glial culture, MCMV (Smith strain) was infected at a multiplicity of infection of 5 or calcium ionophore (A23187; Sigma)27Norris JG Tang L Sparacio SM Benveniste EN Signal transduction pathways mediating astrocyte IL-6 induction by IL-1β and tumor necrosis factor-α.J Immunol. 1994; 152: 841-850PubMed Google Scholar was added in the medium at the concentration of 5 μmol/L. After perfusion and fixation with 4% PFA, the brains were sliced and incubated in X-Gal solution overnight as described above. The slices were embedded in paraffin, sectioned, and subjected to immunohistochemical staining as described below. For the glial cell cultures, the coverslips were fixed in 4% PFA in 0.1 mol/L phosphate buffer at 4°C for 10 minutes and stained with X-Gal. After X-Gal staining, the cultures were post-fixed with 4% PFA in 0.1 mol/L PB. Immunohistochemical staining of sections of X-Gal-reacted brains or coverslips of the cultured glial cells was performed as previously described.25Shinmura Y Aiba-Masago S Kosugi I Li R-Y Baba S Tsutsui Y Differential expression of the immediate-early and early antigens in neuronal and glial cells of developing mouse brains infected with a murine cytomegalovirus.Am J Pathol. 1997; 151: 1331-1340PubMed Google Scholar After pretreatment as described above, the samples were first reacted with rabbit anti-γ-neuron-specific enolase (NSE) antibody (Dr. K. Kato, Aichi Colony, Aich, Japan) for neurons, rabbit anti-glial fibrillary acidic protein antibody (GFAP; Dako Corp., Carpinteria, CA) for astrocytes, rat anti-microglia/macrophage antibody (F4/80; Serotec, Oxford, UK) for microglia, or mouse anti-myelin-associated glycoprotein (MAG; Boehringer Mannheim Biochemica, Mannheim, Germany) for oligodendroglia, incubated with biotinylated secondary antibody and with HRP-conjugated streptavidin, and colored with AEC for brain sections or colored with 3,3′-diaminobenzidine (DAB).25Shinmura Y Aiba-Masago S Kosugi I Li R-Y Baba S Tsutsui Y Differential expression of the immediate-early and early antigens in neuronal and glial cells of developing mouse brains infected with a murine cytomegalovirus.Am J Pathol. 1997; 151: 1331-1340PubMed Google Scholar For the chemiluminescent reporter assay, the Aurora Gal-XE kit was obtained from ICN Pharmaceuticals (Costa Mesa, CA), and a chemiluminescent assay for β-Gal of glial cultures was performed according to the kit's manufacturer. Extracts of glial cells were prepared in the lysate solution of the kit and centrifuged at 12,500 × g for 5 minutes, and 20 μl of supernatant fluid was assayed.28Jain VK Magrath IT A chemiluminescent assay for quantitation of β-galactosidase in the femtogram range: application to quantitation of β-galactosidase in lacZ-transfected cells.Anal Biochem. 1991; 199: 119-124Crossref PubMed Scopus (226) Google Scholar Chemiluminescent emission was measured for 5 seconds using a Lumicounter 700 (Microtech Nichion, Chiba, Japan). Transgenic mice that expressed lacZ gene under transcriptional control of the MCMV-MIE promoter (nucleotides −1343 to −6; Figure 1A) were generated for analysis of the tissue-specific regulation of this promoter. The linearized, purified MCMV-MIEpro1-lacZ transgene was microinjected into pronuclei of fertilized embryos (BDF1BDF1) at the one-cell stage. Founder mice were screened by PCR using tail DNA. PCR amplification was performed in the presence of primers for the transgene or for mouse apoE as an internal control. Several founder mice bearing the MCMV-MIEpro1-lacZ transgene were detected, and three of them are shown in Figure 1B (lanes 1, 3, and 8). Among positive founder mice, three lines were found to express the transgene by X-Gal staining (as shown below). Southern blotting analysis of the three transgenic lines is shown in Figure 1C. BamHI fragment (1.3 kb) including MCMV-MIEpro1 was detected in these transgenic lines after digestion with BamHI (Figure 1C, lanes 4 to 6). Control lanes contained 10 (lane 1) or 1 copy (lane 2) with normal mouse tail DNA. The transgenic lines Tg-1 (lane 4) and Tg-3 (lane 6) had one copy, and the Tg-2 (lane 5) had ∼10 copies. As the transgene (5.1 kb) can be cleaved at one site by NcoI (Figure 1A), the 5.1-kb band was not detected in Tg-1 and Tg-3 after digestion with NcoI, because they had one copy (not shown). The male founder was crossed with nontransgenic C57Bl/c mice and transmitted the gene to ∼50% of the offspring. These hemizygous males were mated with female C57Bl/c, and the offspring were used for experiments. Among several founder mice bearing the MCMV-MIEpro1-lacZ transgene, three lines (Tg-1, -2, and -3) were found to express the transgene by X-Gal staining. The expression pattern in terms of cell specificity was basically the same among the three transgenic lines. As intensity of expression in the brain was the strongest in the Tg-1, cell type specificity of expression of the MCMV MIE promoter was shown using the Tg-1 line. Expression of the transgene in 2-month-old transgenic mice was assayed by either X-Gal or immunohistochemical staining using an antibody specific to β-galactosidase (anti-β-Gal). In the brain, cells positive by either X-Gal staining or β-Gal immunostaining were scattered sparsely in the cerebral cortex (Figure 2A), hippocampus (Figure 2B), cerebellum (Figure 2C), basal ganglia, and brainstem (not shown). The distribution of the positive cells was not in accordance with that of neuronal cells. In the kidneys, strong expression was observed in the epithelial cells of the distal tubules. No staining was observed in the glomeruli or in the proximal tubules (Figure 2D). The salivary glands showed weak expression in a portion of acinar cells (Figure 2E). Only sporadic positive cells were observed in hepatic cells around central veins (Figure 2F) and acinar cells of the pancreas (not shown). Clear expression was observed in the chief cells of the gastric mucosa (Figure 2G). A few positive cells were observed among epithelial cells of the small intestine (Figure 2H), but expression was hardly observed in the large intestine or esophagus (not shown). Lymphoid organs such as the thymus, spleen, and lymph nodes (Figure 1) were negative for expression (Table 1). The spectrum of expression activity of the MCMV IE promoter was much narrower than that of the HCMV IE promoter in transgenic mice reported by Baskar et al16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google Scholar and Schmid et al18Schmid EV Christoph G Zeller R Leder P The cytomegalovirus enhancer: a pan-active control element in transgenic mice.Mol Cell Biol. 1990; 10: 4406-4411PubMed Google Scholar (Table 1). Although positive expression driven by the HCMV IE promoter was reported in the skin, esophagus, testis, spleen, and bone marrow in their transgenic mice, these organs were negative for expression from the MCMV IE promoter (Table 1). Even in the organs positive for MCMV MIEpro1-lacZ expression, only one type of cell was positive in each organ.Table 1MCMV-MIE-lacZ Expression in Transgenic Mice with Reference to HCMV-MIE-lacZ Transgenic MiceMCMVHCMVSystem and organ−1343/−6−670/+54−584/−16Brain Cerebrum+++ Cerebellum+++Gastrointestinal Esophagus−++/− Stomach++/−+/− Small intestine−−+ Colon−−+/− Liver−∼+−− Salivary gland−∼+++/− Pancreas−∼++−Genitourinary Kidney+++ Bladder−++ Testes−++ Ovary−++/−Respiratory Lung−−−Circulatory Heart−∼+++Lymphoid and Blood Spleen−++ Thymus−+/−+/− Bone marrow−+ Blood−−− Lymph node−+/−+Endocrine gland Adrenal gland−+/− Thyroid−+/−+/−Others Smooth muscle−+ Skeletal muscle++Expression activity of MCMV MIE promoter (−1343/−6) was detected by x-Gal staining or immunostaining with anti β-Gal Ab. Expression activity of HCMV MIE promoter (−670/+54) was detected by x-Gal staining or immunostaining with anti β-Gal Ab.16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google ScholarTranscription activity of the HCMV IE promoter (−584/−16) was detected by Northern blotting.18Schmid EV Christoph G Zeller R Leder P The cytomegalovirus enhancer: a pan-active control element in transgenic mice.Mol Cell Biol. 1990; 10: 4406-4411PubMed Google Scholar +, positive; −, negative; −∼+, a portion of cells were positive; +/−, positive or negative. Open table in a new tab Expression activity of MCMV MIE promoter (−1343/−6) was detected by x-Gal staining or immunostaining with anti β-Gal Ab. Expression activity of HCMV MIE promoter (−670/+54) was detected by x-Gal staining or immunostaining with anti β-Gal Ab.16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google ScholarTranscription activity of the HCMV IE promoter (−584/−16) was detected by Northern blotting.18Schmid EV Christoph G Zeller R Leder P The cytomegalovirus enhancer: a pan-active control element in transgenic mice.Mol Cell Biol. 1990; 10: 4406-4411PubMed Google Scholar +, positive; −, negative; −∼+, a portion of cells were positive; +/−, positive or negative. In contrast with the HCMV MIE-promoter-lacZ transgenic mice in which the transgene was intensely expressed in cells morphologically resembling neurons rather than astrocytes, expression of the MCMV MIEpro1-lacZ in transgenic mice was predominantly in glial-like cells but not in neurons as described above (Figure 2, A–C). Furthermore, no expression was observed in the endothelial cells of the brain vessels (not shown), although positive expression was reported in the transgenic mice with HCMV MIE promoter (Table 2). In the cerebellum, positive cells were mainly located in the junction between the molecular and granular layers and were adjacent to the Purkinje cells (arrowheads, Figure 2C); these were considered to be Bergmann glia (arrows). No staining was observed in Purkinje cells or granular cells (Figure 2C).Table 2MCMV-MIEprol-lacZ Expression in Transgenic Mice with Reference to HCMV IE-lacZ Transgenic MiceMCMVHCMVSystem and organ−1343/−6−524/+13−670/+54Cerebrum Neurons−++ Astrocytes+− Oligodendrocytes− Microglia−− Ependymal cells++ Endothelial cells−++Cerebellum Molecular cells−− Purkinje cells−−+ Bergmann cells+ Granular cells−+Expression activity of MCMV-MIE promoter (−1343/−6) was detected by x-Gal staining or immunostaining with anti β-Gal Ab. Expression activity of HCMV-MIE promoter (−524/+13) was detected by x-Gal staining.15Fritschy J-M Brandner S Aguzzi A Koedood M Lüscher B Mitchell PJ Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice.J Neurosci. 1996; 16: 2275-2282Crossref PubMed Google Scholar Expression activity of HCMV MIE promoter (−670/+54) was detected by x-Gal staining or immunostaining with anti β-Gal Ab.16Baskar JF Smith PP Nilaver G Jupp RA Hoffmann S Peffer NJ Tenney DJ Colberg-Poley AM Ghazal P Nelson JA The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.J Virol. 1996; 70: 3207-3214Crossref PubMed Google Scholar +, positive; −, negative.