Engineering of a Mouse for the in Vivo Profiling of Estrogen Receptor Activity
Paolo Ciana,
Giovanni Di Luccio,
Silvia Belcredito,
Giuseppe Pollio,
Elisabetta Vegeto,
Laura Tatangelo,
Cecilia Tiveron and
Adriana Maggi
Institute of Pharmacological Sciences (P.C., G.D.L., S.B.,
G.P., E.V., A.M.) University of Milan 20133 Milan,
Italy
Regina Elena Institute (L.T., C.T.) 00158 Rome,
Italy
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ABSTRACT
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In addition to their well known control of
reproductive functions, estrogens modulate important physiological
processes. The identification of compounds with tissue-selective
activity will lead to new drugs mimicking the beneficial effects of
estrogen on the prevention of osteoporosis and cardiovascular or
neurodegenerative diseases, while avoiding its detrimental
proliferative effects. As an innovative model for the in
vivo identification of new selective estrogen receptor modulators
(SERMs), we engineered a mouse genome to express a luciferase reporter
gene ubiquitously. The constructs for transgenesis consist of the
reporter gene driven by a dimerized estrogen-responsive element (ERE)
and a minimal promoter. Insulator sequences, either matrix attachment
region (MAR) or ß-globin hypersensitive site 4 (HS4), flank the
construct to achieve a generalized, hormoneresponsive luciferase
expression. In the mouse we generated, the reporter expression is
detectable in all 26 tissues examined, but is induced by
17ß-estradiol (E2) only in 15 of them, all
expressing estrogen receptors (ERs). Immunohistochemical studies show
that in the mouse uterus, luciferase and ERs colocalize. In primary
cultures of bone marrow cells explanted from the transgenic mice and
in vivo, luciferase activity accumulates with increasing
E2 concentration. E2
activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen
shows partial agonist activity in liver and bone when administered to
the animals. In the mouse system here illustrated, by biochemical,
immunohistochemical, and pharmacological criteria, luciferase content
reflects ER transcriptional activity and thus represents a novel system
for the study of ER dynamics during physiological fluctuations of
estrogen and for the identification of SERMs or endocrine disruptors.
. At the present time,
molecules active through estrogen receptors (ERs) are used in fertility
control, endocrine dysfunction, and cancer therapy. In postmenopausal
women, estrogen replacement therapy (2) was proven efficacious for the
prevention of osteoporosis (3), and several lines of study suggested
that 17ß-estradiol (E2) has beneficial effects
in cardiovascular (4, 5) and selected neurodegenerative diseases (6).
Unfortunately, the prolonged use of this hormone has been associated
with increased risk of breast and uterine cancer (7). The discovery
that synthetic ligands of the ER may exhibit tissue-specific agonist or
antagonist activity raised a new interest in the use of these compounds
for estrogen replacement therapy (8, 9). These selective estrogen
receptor modulators or SERMs are identified by comparative screening in
cells of different origin to characterize their tissue-specific profile
(agonist/antagonist). Generally, the study is carried out in
transformed cell lines stably or transiently transfected with ER
or
-ß and a reporter of the receptors activated state. In addition to
limiting the analysis to a selected number of cells, this method may
also provide erroneous or defective results. In fact, the
tissue-specific agonist/antagonist activity of SERMs has been
attributed to the presence of cell-specific proteins capable of
interacting with the hormone receptor complex (10), and these proteins
may be aberrantly expressed in cancer cells (11). Thus, the major
shortcoming of this screening procedure is associated with the
requirement of further in vivo analysis for the
identification of the pharmacodynamic properties of the molecule to be
developed. The availability of an engineered mouse carrying an ER
reporter expressed ubiquitously as a transgene would represent a
remarkable advancement for the identification and profiling of new
SERMs. In addition, such a model would be invaluable for the
spatio-temporal localization of ER activity and could provide data of
major impact for the full comprehension of estrogens and ER functions
from development to aging. Such an experimental system can hardly be
generated by classical transgenesis because of the difficulty in
obtaining a regulated expression of the transgene (12). To overcome
this limitation, we made use of insulator sequences previously
described to oppose the interference of the host genome on the
expression of the ectopic genes (13, 14).
In this study we describe a construct that led to ubiquitous and
estrogen-regulated expression of a reporter transgene. The transgenic
mouse we generated represents an innovative model for the study of the
in vivo dynamics of intracellular receptor activity.
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RESULTS
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Generation of the Constructs and Their Preliminary Analysis in
Stably Transfected Cells
The choice of luciferase as reporter gene was dictated by several
factors: 1) no protein structurally related to this enzyme has been
described in mammals; 2) the assay for the quantitation of this
enzymatic activity in tissue homogenates is extremely sensitive; 3)
very efficient antibodies are available for the localization of the
protein by immunohistochemistry. To obtain a minimal constitutive and a
strong estrogen-inducible expression of the reporter, the arrangement
of the promoter cassettes was selected experimentally by transient
transfection studies in MCF-7, SK-N-BE, and HeLa cell lines (not
shown). A number of constructs containing different deletion mutants of
the minimal promoter from the tk gene combined to different
synthetic multimers of the canonical estrogen-responsive element (ERE),
were assayed. The best arrangement found consists of two palindromic
EREs spaced 8 bp apart located at 55 bp upstream from the tk
promoter. To limit position effects and gradual extinction of the
reporter expression (12), we generated constructs in which the
transgene was flanked by either the insulators MAR (matrix attachment
region) (15) or HS4 (ß-globin hypersensitive site 4) (16) (Fig. 1A
). The efficiency of these boundary
elements was tested by stable cotransfection of the constructs
generated, and the pSV2Neo vector in the ER
-positive MCF-7. 48
clones for each construct were isolated, expanded, and tested for
luciferase expression in the presence or absence of 1
nM E2 (Fig. 1B
). In the
absence of hormone, luciferase activity could be measured in 77%
(37/48) and 40% (19/48) of the cells transfected with pHS4 and pMAR,
respectively. In about 80% of these (inducible clones), 16 h of
E2 treatment caused a significant increase in the
reporter activity (at least 3-fold over basal levels). When pERE was
transfected, basal luciferase activity could be detected only in 19%
(9/48) of the clones, and in 44% of these the enzymatic activity was
E2 inducible. Next, we evaluated the relationship
between the number of copies integrated and luciferase expression in
the absence or presence of E2. In the absence of
E2, linear correlation analysis of the two
variables produced lines of best fit with an r coefficient
of 0.66 for pHS4- and 0.79 for pMAR-transfected clones. After 16 h
of E2 induction, the r calculated was
0.47 and 0.54 for the two groups of clones. These values of
r indicate a positive correlation between the two variables
analyzed, even though these data most likely underestimate the
insulator activity because the clones in which transgene rearrangements
had occurred were not eliminated from the analysis.

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Figure 1. Insulator Activity in Stably Transfected MCF-7
Cells
A, Vectors used for the generation of stably transfected cells. B,
MCF-7 cells were cotransfected with pSV2Neo and the indicated
constructs. After selection, 48 single clones for each transfected
plasmid were isolated and expanded. Luciferase activity was measured in
the absence or presence of 1 nM E2 for 16
h. Bars represent the percentage/total of clones
expressing detectable amounts of luciferase (upper
graph) or responsive to the hormonal treatment with at least a
3-fold increase of luciferase activity over basal (lower
graph). The luciferase enzymatic activity was detected in two
separate experiments after triplicate treatment.
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These results are in agreement with previous studies showing that
insulators confer a copy dependency of the transgene expression. In
addition, here we show that these sequences considerably facilitate the
estrogen-regulated expression of the transgene in the chromosomal
context.
Effect of Insulators on Estrogen-Dependent Transcription of the
Reporter Gene in the Mouse
Linearized pMAR and pHS4 vectors deleted of plasmid sequences were
microinjected into oocytes explanted from C57Bl/6xDBA/2
F2 of mice zygotes. This outbred strain was
chosen to ensure a high efficiency of transgenesis (17); furthermore,
the presence of C57BL/6 in the genetic background confers a good
responsiveness to estrogens (18, 19). Seventeen independent lines were
obtained, but only 12 of these were fertile, 9 carrying the pMAR and 3
carrying the pHS4 construct. An initial screening for assessing basal
and estrogen-inducible expression of the luciferase reporter was done
by measuring the reporter enzymatic activity in tissue homogenates from
ovariectomized mice of the F1 generation. Five
organs were initially taken into consideration: uterus, liver and brain
as well known targets for the hormone, and lung and heart as negative
controls. Table 1
shows that among the
lines that integrated the MAR transgene, three showed an
estrogen-inducible expression of the reporter in uterus, brain, liver,
and lung. In line 31 the hormone-inducible expression of the reporter
was found in uterus, liver, and brain, while in lines 56 and 59 it was
restricted to brain. We did not detect any basal or estrogen-inducible
luciferase activity in the heart. In lines 13 and 77, basal expression
of the reporter is low; however, treatment with
E2 did not result in its increase. In transgenic
mice carrying the HS4 construct, we observed very little expression of
the reporter in the organs investigated; only line 61 showed low basal
and E2-induced expression of luciferase.
Considering that minimal promoters are heavily influenced by position
effects, we observed ectopical expression of luciferase in only a few
lines of mice; we concluded that the presence of insulators allows
the position effects to be overcome without interfering
with the hormone-regulated expression of the transgene.
Characterization of Estrogen-Dependent Luciferase Expression in
Transgenic Mice
A further characterization of the activity of the transgene
was carried out in line 2. Luciferase activity was measured in 26
different tissues from 2-month-old female mice, which had been
ovariectomized 2 weeks before the experiment. To verify the capability
of E2 to induce the transgene transcription, mice
were treated for 16 h with either vehicle or
E2 subcutaneously. Figure 2A
shows that in the absence of hormonal
stimulation a considerable level of luciferase expression was found in
tissues such as bone marrow, brain, pituitary, liver, tongue, and
mammary gland, while in others the enzymatic activity found was low, at
the limit of detection. The hormonal treatment induced an increase of
the enzyme content higher than 5-fold with respect to controls in
liver, lung, spleen, bone marrow, brain, and thymus. In eye, uterus,
bladder, skin, adipocyte, and spinal cord, the hormonal treatment
resulted in an accumulation of luciferase less remarkable (between 2.5-
and 4.9-fold over controls), but still clearly visible. Finally, the
treatment did not result in any change in pancreas, tail, aorta,
esophagus, thyroid, stomach, blood, tongue, skeletal muscle, or heart
(Fig. 2B
). When compared with the distribution of ER
and -ß, the
distribution of luciferase activity indicated a strict correlation
between E2 responsiveness and presence of the
hormone receptors. Interestingly, the lung, which was originally taken
as a control ER-negative organ, showed a high responsiveness to the
hormonal treatment. This finding is in line with the recent
report on the high content of ERß in lung (20).

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Figure 2. Localization of Luciferase Activity in Mouse
Tissues before and after E2 Treatment
Adult mice from transgenic line 2 were ovariectomized 2 weeks before
treatment with E2 (50 µg/kg s.c. for 16 h) or
vehicle (vegetable oil). After animals were killed, tissues were
rapidly removed, frozen, and kept at -80 C until assayed. Luciferase
activity measured in tissue extracts is expressed as relative
luciferase units (RLU). A, Basal levels of luciferase activity. B,
Ratio between luciferase activity in estrogen-treated/control mice. The
experiment was repeated three times with a total of six animals per
group. Bars are from a single, representative
experiment.
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Adjacent slices were stained with antibodies raised against ER
or
luciferase (Fig. 3
). ER
immunoreactivity was clearly detected in nuclei of cells in stroma,
endometrium, and glandular epithelium. Cytoplasmic staining of
luciferase was clearly visible in the same cell types. In both
cases, no staining was detected when preimmune serum was used.

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Figure 3. Immunohistochemical Localization of ER and
Luciferase in Uterus of E2-Treated Ovariectomized Mice
ER was immunostained with ER antibodies in nuclei of stroma (S),
lumen (E), and glandular (G) epithelium. No immunoreactivity was
detected with adsorption of the preimmune serum (inset).
Luciferase staining was present in the cytoplasm of cells from stroma,
myometrium, and epithelium lining glands and lumen.
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Bone Marrow Cells in Primary Culture Retain the Estrogen-Inducible
Luciferase Expression
Initial pharmacological characterization of the luciferase
expression was done in primary cultures of bone marrow (Fig. 4
). The cells were treated for 16 h
with increasing concentrations of E2 (0.0110
nM) or with 100 nM of two ER antagonists:
4-hydroxytamoxifen (T) and ICI 182,780 (ICI) alone or in the presence
of 1 nM E2. E2
induced a dose-dependent increase of luciferase accumulation blocked by
the presence of ICI 182,780. ICI 182,780 by itself did not
produce any effect. Conversely, 4-hydroxytamoxifen induced a
significant increase of luciferase levels even though lower than
E2 at the same concentration. In coadministration
with E2, 4-hydroxytamoxifen induced higher
luciferase accumulation, yet the level reached was still lower than
with E2 alone. This is compatible with the
partial agonist activity of 4-hydroxytamoxifen and with the fact that
it is present in the solution at a concentration 100-fold higher than
E2. As control, we also tested progesterone and
dexamethasone (10 nM). Neither ligand had any effect on the
ER reporter (Fig. 4
).

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Figure 4. Expression of Luciferase in Primary Bone Marrow
Cells from Transgenic Mice
Two million bone marrow cells were suspended in phenol red-free RPMI
1640 with 10% stripped serum. Cells were treated with increasing
concentrations of E2 (0.001, 0.01, 1.0, and 10
nM) or with 100 nM ICI 182,780 (ICI) or
4-hydroxytamoxifen (T) alone or with 1 nM E2.
Progesterone (Prog) and dexametasone (Dex) were used at 10
nM final concentration. Control cells (C) were treated with
the same concentration of ethanol present in the hormone solutions
(0.0001%). Bars represent the average ±
SEM of five individual experiments each done in triplicate.
*, P < 0.01 as compared with the control; **,
P < 0.005 as compared with the control; ,
P < 0.05 as compared with the T-treated);
P values were calculated with ANOVA followed by
Scheffé test.
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Taken together, these data confirm that, even in cells explanted from
engineered mice, the transgene is controlled by ligands of ER with
modalities recapitulating those reported for the natural target
genes.
Pharmacological Modulation of Luciferase Expression in
Vivo
Two-month-old male mice were injected s.c. with 50 µg
E2/kg and killed after 3, 6, or 16 h. As
shown in Fig. 5
(upper panel)
the maximal luciferase accumulation was observed at 6 h after
treatment both in liver and bone tissues. When mice were treated for
6 h with increasing concentrations of the hormone (Fig. 5
, middle panel), the maximal effect on luciferase activity was
detected at 50 µg/kg. Interestingly, the administration of 250 µg
E2 /kg induced, in the bone, a luciferase
accumulation lower then with 50 µg/kg. Thus, the luciferase
accumulation is time and dose dependent. Next, the effect of in
vivo administration of the two ER antagonists was investigated.
Figure 5
(lower panel) shows that the s.c administration of
250 µg tamoxifen/kg for 6 h increased the level of luciferase in
liver and bone 12 times and 7 times, respectively, confirming also
in vivo the partial agonist activity of tamoxifen in these
tissues. The injection of 250 µg tamoxifen/kg or ICI 182,780, 1
h before the administration of 50 µg E2/kg,
inhibited the E2-dependent activation of
luciferase expression as expected from the antagonist effect of these
compounds with respect to E2.

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Figure 5. Pharmacological Modulation of Luciferase Activity
in Vivo
Luciferase activity in bone and liver of 2-month-old male mice.
Upper panel, Time course experiments in animals injected
s.c. with 50 µg E2/kg; middle panel, dose
dependency at 6 h treatment; lower panel, blockade
of E2 activation by tamoxifen (T) and ICI 182,780 (ICI) and
partial agonist activity of T. Antagonists (250 µg/kg) were given
1 h before E2. Bars represent the
average ± SEM of five to seven mice. *,
P < 0.01 as compared with the control), ,
P < 0.01 as compared with the
E2-treated); P values were calculated with
ANOVA followed by the Scheffé test.
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DISCUSSION
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We generated a transgenic mouse model for the study of the
dynamics of ER transcriptional activation in primary tissue cultures
and in vivo. Several lines of evidence indicate that the
model generated fulfills its purpose: 1) E2
administration results in accumulation of luciferase in organs and
tissues reported to express either or both of the two ER isoforms; 2)
in uterus, immunohistochemistry shows colocalization of luciferase and
ER immunoreactivity; 3) experiments in primary cultures of bone marrow
show that luciferase activity is controlled by E2
in a dose-dependent fashion (with highest E2
activity compatible with its affinity for the receptors) and ER
antagonists display a profile of activity in line with previous reports
in vivo and in vitro (21); 4) experiments
in vivo show the dose- and time dependency of
E2 activity, the antagonist activity of ICI
182,780, and the partial agonist activity of tamoxifen in bone and
liver.
We believe that the key to the realization of our model was the use of
insulators. It is, in fact, well known that the expression of
transgenes driven by weak promoters is heavily influenced by
enhancers/silencers surrounding the regions of insertion; in addition,
methylation may gradually extinguish their transcriptional activity. In
the past, insulators have been successfully used to counteract these
effects in specific tissues. Here, we demonstrate that their use can be
extended to the achievement of the ubiquitous and regulated expression
of a given gene. Sixty percent of the transgenic lines obtained
expressed luciferase in an estrogen-dependent fashion at least in some
organ. In-depth analysis of one positive line showed that in 26 target
tissues, the expression of the transgene is correctly regulated after
in vivo administration of E2. Yet, in
40% of the mouse lines developed, the expression of luciferase was
either undetectable or not modulated by E2.
This could be due to the use of a weak promoter, which might have
slightly restricted the possibility of reaching detectable levels of
reporter expression. The E2-independent
expression of luciferase may be ascribed to rearrangements of the
vector during the integration in the mouse genome. Indeed, also the
study of stably transfected MCF-7 cells showed that in about 20% of
the clones the expression of luciferase was insensitive to the presence
of E2.
The system generated represents a major advancement for the
understanding of the physiology of compounds active through the ERs. In
the last decade, the ability to transfect cells in culture with
reporters of ER transcriptional activity granted a novel insight into
the complexity of estrogen action. It was shown that the binding of the
hormone-receptor complex to the specific sequences of the promoter is
not sufficient to ensure the hormone-regulated transcription of the
target genes. The ER must, in fact, interact with a series of proteins
modulating its transcriptional activity (22, 23). These findings were
supported by crystallographic studies showing the structural
conformation of ER bound to natural or synthetic ligands (24, 25).
These and other investigations on steroid receptors demonstrated how
synthetic ligands, by inducing specific structural conformations that
modify the possibilities of the receptor to interact with its
coregulators, may change its transcriptional activity in a
tissuespecific fashion (10, 23, 26). In addition, several studies
underlined that the binding of the specific hormone is not
indispensable to ER transcriptional activation. Unliganded ER was shown
to regulate the transcription of target genes after activation of
specific kinases (27, 28). Finally, ER dosage may also constitute an
important element in the control of ER tissue-specific activities.
A major challenge at present is to demonstrate how these mechanisms are
relevant in physiological systems and how ER activity is regulated in
its numerous target cells. The model generated will facilitate these
studies by providing a system in which the activity of the receptor on
ERE-containing genes can be assessed in a very restricted time frame.
To this aim, we purposely made use of the natural firefly luciferase
gene, the turnover of which in mammalian cells is about 3 h (29).
By measuring the levels of luciferase accumulation, therefore, we will
be able to monitor the state of activity of the receptor in response to
the fluctuating hormone levels during the estrous cycle or after
administration of ER ligands. In addition, these mice will allow
identification of novel tissues and cell types targeted for the
hormone in vivo in both sexes.
Further investigation is necessary to understand whether the high
content of luciferase in bone marrow, brain, tail, tongue, or liver
observed in this study should be ascribed to the tissue characteristics
facilitating the recovery/measurement of the luciferase enzymatic
activity, to the low catabolism of the exogenous protein, or to the
activation of the unliganded resident receptor via cross-coupling with
membrane receptors. We would rule out the possibility of luciferase
induction by ERR (ER-related receptor) orphan receptors based on the
observation that tissues such as kidney and heart, known to express
very high concentrations of ERR
and
, display a very low basal
activity of the reporter (30, 31, 32). Similarly, a more accurate
evaluation of the time course of E2 induction is
necessary before drawing any conclusion on the potency of the hormonal
treatment in the various organs. The present study was carried out at
16 h of hormonal treatment. It is likely that the relatively low
E2-dependent accumulation of luciferase that we
observed in certain organs (e.g. uterus, mammary glands) is
due to ER down-regulation, which in these organs occurs in a few hours
after E2 administration. Appropriate time-course
studies will better clarify the kinetics of ER activity in the various
tissues.
From the pharmacological point of view, the system generated is very
interesting, particularly for the identification of novel SERMs because
it will identify in which organs the molecule of interest
displays full, partial agonist, or antagonist activity. The preliminary
assessment of the activity of 4-hydroxytamoxifen in bone marrow cells
and in vivo, supports the validity of this model in this
type of studies. In previous studies, reporter-based systems for the
in vivo identification of ligands for intracellular
receptors were generated using fusion proteins between the RXR and RAR
ligand binding domain and the DNA binding domain of the yeast protein
GAL4 (33, 34). These systems were proved to be useful for the detection
of endogenous ligands; however, because of the relevance of
protein/protein interaction in the activity of intracellular receptors,
GAL4-receptors fusion products might be unable to undergo
conformational changes indispensable for the action of tissue
selective synthetic ligands (10, 23, 26).
In spite of the fact that the system generated will not provide an
insight on the exact nature of the ER activated [ER
, ERß, or
other proteins not known active through estrogen response elements
(EREs)], appropriate breeding of the ERE transgenic mice with
selective ER
and -ß knockout (K.O.) mice will erase this
limitation.
The transgenic mouse generated in this study can be used to produce
models for the physiological, pharmacological, and toxicological
analysis of other intracellular receptors. In addition, because of the
intrinsic characteristics of the reporter above specified, the model
could be particularly suited for studying the pharmacokinetic profile
of natural and synthetic ER ligands. Finally, these transgenic mice can
be used as biosensors to investigate whether environmental or food
pollutants act as endocrine disruptors by interfering with the
physiological state of ER activity.
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MATERIALS AND METHODS
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Plasmid Construction
Each functional cassette of the vector used for transgenesis was
flanked with unique restriction sites to facilitate further
manipulations. Each element of the construct generated was sequentially
cloned in the vector pBluescript (Stratagene, La Jolla,
CA). The basic construct without insulators was named pERE and contains
two canonical EREs (35) (ERE2X) spaced by 8 bp, a minimal
thymidine kinase (tk) promoter from herpes
simplex virus (36) (55 bp downstream from the ERE2X) and the
luciferase reporter gene (Fig. 1A
). This construct was
assembled with the following components: 1) the 2,731-bp DNA fragment
encoding the luciferase excised from the pGl2basic vector
(Promega Corp., Madison, WI) with the SalI
restriction enzyme blunted and ligated into the blunted
HindIII site of pBluescript; 2) the 168-bp
BamHI/XhoI fragment containing the tk
promoter from pBLCAT2 (37), blunted and ligated into the blunted
PstI site of the pBluescript; 3) the 82-bp
XhoI/ClaI fragment containing the ERE2X excised
from pGL2basic vector (Promega Corp.) in which it has been
previously cloned (see below), blunted, and ligated into the blunted
SalI site of the pBluescript.
Two tandem copies of the insulators HS4 (2.4-kb DNA fragment) from
chicken ß-globin gene were obtained by digesting the
vector pBS(II)HS4, generously provided by S. Y. Tsai (38), with
SalI restriction enzyme; a single copy of MAR (3-kb DNA
fragment) from chicken lysozyme gene was excised with
digestion of the pBSKMAR, kindly provided by L. Hennighausen (39), by
XbaI/BamHI restriction enzymes. The insulator
fragments were blunted and inserted in the blunted KpnI and
NotI sites located at the 5'- and 3'-end of pERE, giving the
pHS4 (EMBL accession no. AJ2777959) and pMAR (EMBL accession no.
AJ2777959) constructs.
Generation of ERE2X
The two oligonucleotides, 5'-GATCCGCAGGTCACAGTGAC CTA-3' and
5'-GATCTAGGTCACTGTGACCTGCG-3', were annealed, the resulting double
strand oligo was ligated and digested with BamHI, and the
bands corresponding to monomers or multimers were extracted from an
acrylamide gel as described previously (40) and ligated into the
BglII site of pGL2basic vector.
Cell Cultures and Transfections
Breast carcinoma MCF-7, neuroblastoma SK-NBE, and cervix
carcinoma HeLa cell lines were routinely grown in RPMI 1640 medium
supplemented with 10% FBS. Stable transfections of MCF-7 cells were
performed with the calcium phosphate procedure as previously described
(41). Twenty four hours before transfection, 1.5 x
106 cells were seeded in Petri dishes with RPMI
1640 supplemented with 10% FCS; 6 h before addition of 1 ml
CaPO4/DNA mixture the medium was replaced with
DMEM supplemented with 10% FCS. The CaPO4/DNA
mixture used for transfection contained 1 ng of pSV2Neo plasmid
expressing G418 resistance gene (CLONTECH Laboratories, Inc., Palo Alto, CA; GenBank accession no. U02434) together with
10 µg of the pERE, pHS4, or pMAR vectors and 9 µg salmon sperm DNA.
Forty eight hours after transfection, 300 µg/ml G418 (Life Technologies, Inc.) were added to the culture medium. Medium and
selective agents were replaced three times a week. After 21 days
selection, 48 clones for each transfection were isolated with cloning
rings and expanded. To test the expression of luciferase, each clone
was grown in RPMI 1640 without phenol red and supplemented with 10%
dextran charcoal-stripped FBS (DCC-FBS) (41) for at least 1 week
before 16 h induction with E2 (1
nM in 0.00001% ethanol). Control cells were treated for
24 h with 0.00001% ethanol. Protein extracts were obtained as
previously described (27), and the enzymatic assay was carried out as
described in detail below.
Transgenic Mice
For microinjection, linearized pMAR and pHS4 constructs depleted
of plasmid sequences were obtained with BsshII restriction
enzyme digestion. With these vectors two different types of transgenic
mice were produced by pronuclear DNA injection of zygotes
C57Bl/6xDBA/2, F2 generation, using standard
procedures (17). Injected zygotes were reimplanted into pseudopregnant
B6D2F1 (C57Bl/6xDBA/2) foster mothers to complete their development.
Genomic DNA was extracted as previously described (42) from tail
biopsies and used for genotyping. Briefly, tissues were lysed by
addition of 1% SDS, 50 mM Tris-HCl, pH 8, and
200 µg/ml Proteinase K and incubation overnight at 37 C; DNA was then
purified by phenol extraction and ethanol precipitation. DNAs from the
founders and their littermates were screened by PCR analysis. PCR
amplification was carried out in a buffer containing 10
mM Tris-HCl (pH 8.0), 50 mM
KCl, 1.5 mM MgCl2, 0.2
mM deoxynucleotide triphosphates, 0.25
µM of each primer, and 2 U of TAQ polymerase
for 1 µg genomic DNA template. The primers used were
5'-GGCAGAAGCTATGAAACGAT-3' and 5'-CGACTGAAATCCCTGGTAAT-3'; after 30
cycles (30 sec at 95 C, 30 sec at 55 C, and 30 sec at 72 C) the
products were analyzed on 2.5% agarose gels stained with ethidium
bromide. At the third week of age, all the potential founders obtained
from pHS4 and pMAR microinjection were screened by PCR. From the pMAR
and pHS4 groups, 10 and 7 individuals, respectively, were identified as
positives for the presence of the transgene. For the experiments we
used heterozygous littermates obtained by mating our founders with
B6D2F1 wild-type mice.
Heterozygous female mice (2 months old) were ovariectomized and after 2
weeks injected s.c. with 50 µg E2/kg or with
vehicle (vegetable oil) as control. Sixteen hours later the animals
were killed, and the tissues were dissected and immediately frozen on
dry ice. For the in vivo pharmacological studies with ER
antagonists, 2-month-old heterozygous male mice were treated by s.c.
injections of the different compounds dissolved in vegetable
oil. Tissue extracts were prepared by homogenization in 500 µl of 100
mM KPO4 lysis buffer (pH
7.8) containing 1 mM dithiothreitol, 4
mM EGTA, 4 mM EDTA, 0.7
mM phenylmethylsulfonyl fluoride, three cycles of
freezing-thawing, and 30 min of microfuge centrifugation at maximum
speed. Supernatants, containing luciferase, were collected and protein
concentration was determined by Bradfords assay (43).
Luciferase Enzymatic Assay
Luciferase enzymatic activity in the cell and tissue extracts
was measured by a commercial kit (luciferase assay system,
Promega Corp.) according to the supplier indications. The
light intensity was measured with a luminometer (Lumat LB 9501/16,
Berthold, Wildbad, Germany) over 10 sec and expressed as relative light
units (RLU) over 10 sec/µg proteins.
Immunohistochemistry
Uteri of ovariectomized mice, treated as before, were dissected
and fixed through immersion in 4% paraformaldehyde in 0.1
M phosphate buffer pH 7.2 (PB), for 5 h. Tissues were
dehydrated with an ascending ethanol scale, clarified with xylene, and
processed for paraffin embedding. Serial 4 µm microtome sections were
cut and collected onto slides coated with poly-L-lysine
(Sigma, St. Louis, MO). After 16 h drying at 37 C,
sections were hydrated through a descending ethanol scale and boiled in
10 mM citrate buffer (pH 6.0) for 15 min in a microwave
oven, washed for 10 min with PBS, and then processed for luciferase and
ER
immunodetection at room temperature. Sections were first
incubated for 30 min with 0.3%
H2O2 to quench endogenous
peroxidase activity and subsequently washed three times with PBS for 10
min. After saturation with 10% preimmune goat serum
supplemented with 0.3% Tween 20 (Sigma), sections
were incubated with the antiluciferase (Sigma, 1:1800
dilution in PBS with 10% goat serum and 0.3% Tween 20) or anti-ER
(kindly provided by J. Green, 5 µg/ml in 10% goat serum and 0.3%
Tween 20) polyclonal antibodies for 16 h, washed with PBS (six
times, 10 min each), incubated for 60 min with an antirabbit secondary
antibody (raised in goat, 1:200 dilution in PBS supplemented by 1%
goat serum and 0.3% Tween 20; Vector Laboratories, Inc.,
Burlingame, CA) and then washed again (six times with PBS, 10 min
each). Antibody-antigen detection was obtained by 40 min incubation
with avidin-biotin-horseradish peroxidase (HRP) from an ABC kit
(Vector Laboratories, Inc.). Immunostaining was visualized
by exposure to HRP substrate 3,3'-diaminobenzidine (DAB Fast, Tablet
Set, Sigma). After one wash in PBS and few tap water
changes, sections were allowed to air dry and then covered. Pictures
were taken with a digital camera (Coolpix 990; Nikon,
Melville, NY) applied to a Axioscope microscope (Carl Zeiss, Thornwood, NY).
Primary Bone Marrow Culture
After mice were killed, bone marrow cells were flushed out from
femur and tibia of ovariectomized animals, using a syringe filled with
PBS. Cells were collected in a 15 ml Falcon tube (Becton Dickinson and Co., Meylan Cedex, France) and washed once with
PBS. After centrifugation the cell pellet was resuspended in RPMI 1640
supplemented with 10% DCC-FBS; cells were counted and plated in a
six-well dish (2 x 106cells per well). For
the treatment, all compounds were dissolved in ethanol and added to the
medium at the indicated concentration. After 16 h the cells were
collected in Eppendorf tubes, washed once with PBS,
re-suspended in 100 µl KPO4 lysis buffer (as
above), and frozen and thawed three times. After 30 min microfuge
centrifugation at maximum speed, supernatants were collected for the
determination of protein concentration and luciferase activity.
Experimental Animals
Animal experiments performed in this study were conducted
according to the "Guidelines for Care and Use of Experimental
Animals."
 |
ACKNOWLEDGMENTS
|
---|
We thank Laura Pozzi for her experimental advice and Monica
Rebecchi and Clara Meda for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Prof. Adriana Maggi, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: adriana.maggi{at}unimi.it
This study was supported by the European Community Program BIOMED
(Grant BMH4-CT972286); Telethon (Grant E.600); Italian Association
for Cancer Research (AIRC); and CNR Targeted Project
Biotechnology, Murst 40%.
Received for publication December 4, 2000.
Revision received February 28, 2001.
Accepted for publication March 19, 2001.
 |
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