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From the Second Department of Pathology*
and
Animal Research Center,
Hamamatsu University
School of Medicine, Hamamatsu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.14-18 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.19,20 MCMV (Smith strain) DNA21 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 ß-galactosidase (ß-Gal) coding sequence, and poly(A) and intron from mouse protamine, provided by Dr. R. D. Palmiter, University of Washington.22 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.
Generation of Transgenic Mice
Transgenic mice were produced by standard
techniques.23
Purified linearized DNA of the
MCMV-MIEpro1-lacZ DNA was injected into the pronuclei of fertilized ova
derived from BDF1 (C57BL/6 female x 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.24
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.
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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-ß-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.22 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-ß-galactosidase antibody (anti-ß-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).25
Primary Glial Cell Cultures
Primary cortical glial cell cultures were prepared according to the methods of Togashi et al26 and Norris et al27 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 x 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)27 was added in the medium at the concentration of 5 µmol/L.
Combination of X-Gal Staining and Immunohistochemical Staining
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.25
After pretreatment as described above, the samples were first reacted
with rabbit anti-
-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).25
Chemiluminescent Assay of ß-Galactosidase
For the chemiluminescent reporter assay, the Aurora Gal-XE kit was obtained from ICN Pharmaceuticals (Costa Mesa, CA), and a chemiluminescent assay for ß-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 x g for 5 minutes, and 20 µl of supernatant fluid was assayed.28 Chemiluminescent emission was measured for 5 seconds using a Lumicounter 700 (Microtech Nichion, Chiba, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
Cell Type-Specific Expression in Transgenic Mice
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 ß-galactosidase
(anti-ß-Gal). In the brain, cells positive by either X-Gal staining
or ß-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
al16
and Schmid et al18
(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.
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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)
.
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To further characterize the glial cells expressing MCMV
MIE-promoter-lacZ, the positive cells were double stained
immunohistochemically for neuronal, glial, oligodendroglial, and
microglial markers. After X-Gal staining of whole brains or brain
slices, paraffin-embedded or frozen sections were subjected to
immunohistochemical staining for cell type markers. The lacZ-expressing
cells were not double stained with anti-NSE antibody (Ab) (Figure 3A)
but partially double stained with
anti-GFAP Ab (Figure 3, B and C)
, probably because only a portion of
astrocytes may be stained by anti-GFAP Ab; fibrous astrocytes in the
upper cerebral layers (Figure 3B)
and the white matter (Figure 3C)
were
stained with anti-GFAP Ab, but protoplasmic astrocytes were not
stained.29
It was difficult to define the cell types by
staining for oligodendroglial and microglial markers in brain sections.
Therefore, primary culture of the glial cells from transgenic mice with
MCMV-promoter-lacZ was performed. More than 50% of ß-Gal-positive
cells were double stained with anti-GFAP Ab (Figure 3D)
. Microglial
cells detected by staining with the F4/80 Ab were not double stained
with X-Gal (Figure 3E)
. Relatively fewer oligodendroglia in the
cultures were stained with anti-AMS Ab, but these cells were not double
stained with X-gal (Figure 3F)
. These results suggested that the MCMV
MIE promoter in our transgenic mice showed astrocyte-specific
expression in the brain.
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Glial cultures from the transgenic mice cultured in 12-well plates
were infected with MCMV (Smith strain) at a multiplicity of infection
of 5. MCMV MIE promoter activity measured by chemiluminescent assay for
ß-Gal was significantly stimulated by viral infection when compared
with that of uninfected cultures (Figure 4A)
. As HCMV MIE promoter was reported to
be stimulated via induction of NF-
B,30
calcium
ionophore (A23187)27
was added in the culture medium (5
mol/L). MCMV MIE promoter activity was also significantly stimulated
(Figure 4B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
There are two possible explanations for the more specific expression
activity of the MCMV IE than the HCMV IE promoter reported recently in
transgenic mice.15,16
Host cellular transcription factors
may interact with the MCMV MIE promoter more naturally in transgenic
mice because of the homogeneity of this combination, although
transcription factors are thought to be evolutionarily well preserved
across the species.33
Alternatively, these observations may
have been because the MCMV MIE promoter region used here was 1338 bp in
length (-1343 to -6), far longer than the HCMV IE promoter used in
previous transgenic studies (-524/+1314,15
and
-670/+54.16,17
It has been reported that the HCMV MIE
promoter may contain a modulator region located between nucleotides 750
and -1145 of the the HCMV MIE promoter region that negatively
regulates expression of the major IE gene in undifferentiated cells but
positively influences expression in differentiated
cells.13,34
Although whether there is a similar modulator
region in the MCMV MIE promoter has not been determined, it is possible
that the MCMV MIE promoter used in the present study contained such a
region. It is noteworthy that expression of the MCMV MIEpro1-lacZ in the brain of the transgenic mice in the present study was predominantly observed in glial cells. According to the combination of X-Gal staining and immunostaining for cell markers, the positive cells were astrocytes, because they were positive for GFAP but negative for neuronal, oligodendroglial, and microglial markers. This result was in contrast with the expression pattern of the HCMV MIE promoter-lacZ used in previous transgenic studies, which was observed in neurons and endothelial cells but not astrocytes.15,16
Interestingly, the MCMV MIE promoter-lacZ was expressed only in the Bergmann glia in the cerebellum in the present study based on the specific distribution of the cells adjacent to Purkinje cells. The transcription factors binding to the cAMP response element (CRE)-binding protein (CREB)-1 are constitutively expressed in periventricular glia and Bergmann glia.35,36 It was also shown that the major enhancer of HCMV can be regulated by cAMP treatment in a cell-type-specific manner,37 and the sequence for the CREB was detected between nucleotides -78 and -56 of the HCMV MIE promoter (UL112/113),38 corresponding to the region shown in MCMV.39
The cell type specificity of MCMV MIE promoter-lacZ expression in the transgenic mice was consistent with target cells of HCMV infection in various organs such as the brain, kidneys, stomach, salivary glands, and pancreas,2-4,40 although the levels of expression in the latter two organs were low. Interestingly, astrocyte-specific expression of the MCMV MIE pro1-lacZ constructs in the transgenic mouse brain was consistent with the observation that these cells are among the targets in HCMV encephalitis in humans.41 We have previously shown that the glial cells expressing GFAP expressed the major IE 89-kd antigen in the neonatal mouse brain after intrauterine infection, and the glial cells were preferentially transferred to lytic infection with expression of the late gene,25 although the viral early nuclear antigen was expressed in various mesodermal cell types in MCMV-infected embryos42 and neuronal cells in the postnatal mouse brain.43
Unexpectedly, we did not detect expression of the MCMV MIE promoter-lacZ in the lung, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, or bone marrow, although these organs are known to be susceptible to HCMV infection. To explain the lack of MIE promoter activity in the susceptible organs, Baskar et al16 suggested that expression may be below the limits of detection, this promoter may require other viral or virally modified host factors, or the cell may revert back to fetal type in these organs. In addition, it is possible that some factors may suppress the MIE promoter activity in these organs, resulting in latent infection, because latent infection has been reported in the these organs, including the salivary glands.44-47
The MCMV MIE promoter was stimulated in the glial cultures from the
transgenic mice by infection with MCMV or by adding calcium ionophore
in culture medium, although the stimulation was not extensive. Calcium
ionophore was reported to activate NF-B via phosphorylation of
protein kinase C.27
HCMV IE promoter was stimulated by
induction of the NF-B.30
Conversely, activity of NF-B
was stimulated by infection with MCMV48
and with
HCMV.49
However, HCMV IE gene products were involved in
negative autoregulation of the HCMV IE promoter.50
Further
investigation is to be scheduled for the mechanism of the stimulation
of the transgene in glial culture.
In conclusion, we have shown that expression activity of the MCMV MIE promoter (-1343 to -6) is highly cell type specific in transgenic mice. The spectrum of organs and cells positive for the activity was narrower than that of the HCMV MIE promoters previously reported in transgenic mice. In the brain, expression activity of the promoter was restricted to astrocytes with no expression in neurons, oligodendroglia, microglia, or endothelial cells.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant (8A-8–23) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan.
Accepted for publication December 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity and immunologic (type II) nitric oxide synthase expression. Proc Natl Acad Sci 1997, 94:2676-2680
. J Immunol 1994, 152:841-850[Abstract]
is mediated via induction of NF-B. Virology 1995, 208:197-206[Medline]
inhibits the murine cytomegalovirus immediate-early gene expression by down-regulating NF-B activity. Virology 1995, 211:251-260[Medline]
B activity by transactivating the NF-B p105/p50 and p65 promoters. J Virol 1995, 69:5391-5400[Abstract]
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