Conditional Liver-specific Expression of Simian Virus 40 T Antigen Leads to Regulatable Development of Hepatic
Neoplasm in Transgenic Mice*
Elanchezhiyan
Manickan
,
Jujin
Satoi
,
Timothy C.
Wang§, and
T. Jake
Liang
¶
From the
Liver Diseases Section, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892 and the
§ Gastrointestinal Unit, Department of Medicine,
Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, October 26, 2000, and in revised form, January 12, 2001
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ABSTRACT |
Adaptive epigenetic changes and toxicity often
accompany constitutive expression of a transgene or knockout of an
endogenous gene in mice. These considerations potentially limit the
usefulness of transgenic technology in studying the in vivo
functions of a gene. Using conditional gene expression technology, it
is possible to override such restrictions to achieve temporal and
tissue-specific manipulation of gene expression in vivo.
Based on the tetracycline regulatory system, we established a binary
transgenic model in which the conditional expression of two transgenes,
SV40 T antigen (TAg) and lacZ, can be tightly regulated in
the liver by administration of tetracycline. The mouse albumin or mouse
major urinary protein promoter was used to achieve
liver-specific expression of the tetracycline-responsive
transcriptional activator (tTA) in one set of transgenic mice. These
mice were crossed with transgenic mice carrying either TAg or
lacZ under the control of the tTA-regulated promoter.
Analyses of mice transgenic for both tTA and TAg (or lacZ)
revealed that the liver-specific expression of the transgenes could be
suppressed to undetectable levels and regulated in a reversible fashion
by tetracycline administration and withdrawal. Mice with tTA and TAg
transgenes developed hepatocellular adenomas and hyperplasia that could
be prevented by continuous tetracycline administration. Our report
demonstrates the value of this binary transgenic model in studying the
physiological functions of any potential genes of interest in a
liver-specific manner.
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INTRODUCTION |
Transgenic mouse models have been used widely to study gene
expression under in vivo physiological conditions. With
tissue-specific promoters, it is possible to target gene expression
exclusively in a specific tissue such as liver. However, constitutive
expression of a transgene, especially if it deleteriously affects the
tissues to which it is targeted, often results in prenatal or postnatal death or causes a variety of compensatory changes (1) in the overall
gene expression pattern of the tissue. These changes could result in
unexpected phenotypes that may not reflect the true biological
functions of the transgene (2-5). In certain situations, the transgene
is silenced by epigenetic events such as methylation because of its
adverse effects on the cells (2, 6). Furthermore, the transgene behaves
as a self-antigen, inducing negative selection of reactive T cells in
the thymus and causing the animals to become immunologically tolerant
to the transgene. Therefore, the immunologic response to the
transgene cannot be easily studied without special manipulation. This
aspect is particularly relevant in models of autoimmune or viral disease.
To circumvent these problems, it is necessary to develop a system by
which the expression of a transgene can be induced at desired time
points and otherwise be kept completely silent for an extended period
of time. Such a model may also allow a viral or self-antigen to
escape the thymic selection process so the immunologic response can be
studied. The use of inducible promoters, such as heat shock,
metallothionein, and murine mammary tumor virus promoters, that can be
regulated by temperature, zinc, or dexamethasone is frequently
associated with a high basal level of expression, a less than
impressive induction of the transgene, a relative lack of specificity,
and a possible toxicity of the induction method (3, 4, 7). Conditional
gene expression in vivo has been achieved using a variety of
model systems. One of them takes advantage of the
cre-lox recombination system by which a transgene
can be activated and an endogenous gene deleted in a tissue-specific
and time-dependent manner (8). However, this system
requires the exogenous delivery of the cre gene (usually by
an adeno- or retrovirus), and the induction is irreversible. Recently
several drug- or ligand-inducible systems have been developed in
vitro and, to some extent, in vivo (7). These systems
involve the use of a chimeric transcriptional activator that reversibly activates a target gene in response to the administration of the inducing agent. One of the systems that uses the intrinsic properties of the Escherichia coli tetracycline resistance operon has
been applied widely to the generation of cell lines with tightly
regulated gene expression in response to tetracycline (4). This system has also been applied to the transgenic models of lymphoid and salivary
development (5), but no such model has been reported for liver-specific
expression. In this study, we developed a binary transgenic mouse model
that permits the tight control of transgene expression in a temporal
and liver-specific fashion by tetracycline (the tetracycline-repressed
regulatable system). We evaluated the efficiency of this system in
transgenic mouse strains carrying one of two reporter genes, one coding
for SV40 T antigen (TAg)1 and
the other for
-galactosidase (lacZ), and the
tetracycline-responsive transcriptional activator (tTA) under the
control of liver-specific promoter of mouse albumin (Alb) (9, 10) or
mouse major urinary protein (MUP) (11, 12). tTA, in the absence of
tetracycline, activates the reporter genes through binding to the
reporter promoter sequence.
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MATERIALS AND METHODS |
Generation of Transgenic Mouse Lines--
The transgenes
containing tTA under the control of the mouse albumin (Alb-tTA) or
mouse major urinary protein (MUP-tTA) promoter were constructed (see
Fig. 1A). For construction of the Alb-tTA transgene, the
EcoRI-BamHI fragment containing the tTA sequence from pUHD15.1 (22) was cloned into the EcoRI and
BamHI sites of the pBSIIKS+ vector (Stratagene).
The tTA fragment released by digestion of the resulting plasmid with
NotI and XhoI was inserted into the pGEMAlbSVtpA
vector (33). The resulting construct was digested with NheI
and NsiI sites to generate the Alb-tTA transgene (~4.5 kilobase pairs). The MUP-tTA transgenic construct was generated the
following way. The PstI-KpnI fragment containing
the MUP promoter from MUPII (11) was inserted into the NsiI
and KpnI sites of pSVSPORT1 (Life Technologies, Inc.),
replacing the SV40 promoter. The EcoRI-HpaI
fragment containing tTA and SV40tIVSpA from pUHD15.1 was inserted into
the SalI (treated with Klenow for blunt end) and
EcoRI sites of the previous construct. The resulting plasmid was then digested with ClaI and MluI to generate
the MUP-tTA transgenic fragment (~4.0 kilobase pairs). These
transgenes were introduced into fertilized eggs of FVB/n mice using
standard transgenic techniques (13). Mice transgenic for SV40 T antigen
(tetO-TAg; from Shimon Efrat, Albert Einstein College of
Medicine, Bronx, NY) (14) or
-galactosidase (lacZ; from
the Jackson Laboratory, Bar Harbor, ME) (15) linked to the
cytomegalovirus minimal promoter fused to the tTA-binding sequences
(tetO) have been described previously. Genomic DNA isolated
from tail clips of the offspring was used for genotyping. Genotyping of
the Alb-tTA and MUP-tTA transgenic mice was performed using standard
Southern blotting, and genotyping of the TAg mice was performed using
the polymerase chain reaction (PCR) with TAg-specific primers (sense,
5'-GGA ATA GTC ACC ATG AAT GAG TAC AG-3'; and antisense, 5'-GGA CAA ACC
ACA ACT AGA ATG CAG TG-3').
Administration of Tetracycline--
Slow-release tetracycline
pellets (Innovative Research of America, Sarasota, FL) were implanted
subcutaneously in the shoulder regions of mice using a trochar
as described by the manufacturer. These pellets release 0.7 mg of
tetracycline hydrochloride/day for 21 days. As negative controls,
placebo tablets obtained from the same manufacturer were similarly
implanted. Expression of the transgene was measured on day 20. A group
of mice were killed and analyzed for their transgene expression on day
28 (7 days after the dissolution of the tetracycline pellets) to
evaluate the reversibility of the transgene expression.
Protein and RNA Extraction--
100 mg of liver or spleen tissue
from the transgenic animals was homogenized in buffer containing 1%
Nonidet P-40, 1% sodium deoxycholate, 1% SDS, 150 mM
sodium chloride, 10 mM sodium phosphate, 2 mM
EDTA, 50 mM sodium fluoride, and protease inhibitor mixture (Pefabloc SC, Roche Molecular Biochemicals) at a concentration of 1 mg/ml. Homogenates were centrifuged at 15,000 × g for
20 min at 4 °C, and the supernatants were collected. Total protein concentrations of the lysates were measured using a Coomassie Plus
protein assay reagent kit (Pierce). Total RNAs from liver, spleen,
brain, tongue, kidney, lung, heart, ovary, skin, and skeletal muscle
were isolated using the Ultraspec-II RNA isolation reagent (Biotecx
Laboratories, Houston, TX). Briefly, 100 mg of tissue was homogenized
using a glass homogenizer in 1 ml of Ultraspec-II RNA isolation
reagent. The RNA was precipitated with isopropyl alcohol and dissolved
in diethyl pyrocarbonate-treated water.
Analysis of T Antigen and lacZ Expression--
T antigen was
detected by Western blotting using mouse monoclonal anti-TAg antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) and peroxidase-labeled goat
anti-mouse IgG as the secondary antibody (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD) with a chemiluminescence
detection kit (Santa Cruz Biotechnology). The actin level of each
sample was tested as an internal protein control. Reverse transcription
was performed using the Thermoscript RT-PCR system kit (Life
Technologies, Inc.). Briefly, 5 µg of total RNA was mixed with
TAg-specific antisense primer (10 µM), and the volume was
adjusted to 10 µl using diethyl pyrocarbonate-treated water. The
RNA/primer mixture was incubated at 65 °C for 5 min. 10 µl of the
reverse transcription mixture was added to the RNA/primer mixture, and
the reaction was continued at 50 °C for 1 h and terminated by
heating at 85 °C for 5 min. 1 µl of RNase H was added for an additional 20 min at 37 °C. Synthesized cDNA was amplified by a
standard PCR protocol: one cycle at 94 °C for 4 min; 35 cycles at
94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1.5 min; and a
final cycle at 72 °C for 5 min. PCR-amplified fragments were electrophoresed on 1% agarose gel and analyzed by Southern blotting using 32P-labeled TAg cDNA probe.
-Galactosidase activity in the liver or spleen lysate was measured
using the Galacto-light
-galactosidase detection kit (Tropix Inc.,
Bedford, MA). Briefly, 100 mg of liver or spleen tissue was homogenized
in 1 ml of lysis solution containing 10 µl of a 0.1 M
solution of dithiothreitol, 1 µl of 30% hydrogen peroxide, and 100 µl of protease inhibitor mixture (Complete, Roche Molecular
Biochemicals; from a stock solution containing one tablet in 1 ml of
water). The protein concentration of the lysate was determined, and 100 µg of lysate was used for quantitation of
-galactosidase activity.
Recombinant
-galactosidase (Sigma) was used as a positive control,
and liver lysate from nontransgenic mice was used as a negative
control. The final reaction mixture containing light-emitting
substrates was measured using a plate luminometer (Packard Instrument
Co.), and the results of the
-galactosidase activity were calculated
as counts/min.
Analysis of Tumor Development in TAg Mice--
Offspring of the
cross between Alb-tTA line 3123 and tetO-TAg were subjected
to tetracycline or placebo treatment continuously for a period of 12 months. These mice were killed at the end of the follow-up period.
Livers and other organs were examined for any gross morphological
changes. A piece of each tissue was fixed in buffered Formalin, and
8-µm-thick sections were cut. Sections were stained with standard
hematoxylin/eosin and evaluated for any histological changes.
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RESULTS |
Development of a Binary Transgenic System--
Two Alb-tTA (lines
3140 and 3123) and three MUP-tTA (lines 7552, 7545, and 7016)
transgenic lines were generated. Expression of tTA could be detected by
RT-PCR in the livers from all lines (data not shown). The heterozygous
animals of each tTA line were crossed with the heterozygous
tetO-TAg or tetO-lacZ mice to generate double-positive, single-positive, or double-negative transgenic mice
for the experiment. Alb-TAg line 3140 was generated by crossing Alb-tTA
3140 mice with tetO-TAg mice, and Alb-TAg line 3123 was generated by crossing Alb-tTA 3123 mice with tetO-TAg mice.
The Alb-lacZ mice were generated the same way. MUP-TAg and
MUP-lacZ lines 7552, 7545, and 7016 were generated by
crossing each MUP-tTA line with tetO-TAg and
tetO-lacZ, respectively.
Animals obtained from each cross were divided into three groups based
on the genotype, viz. double-positive animals (which contain
both tTA and TAg transgenes), tTA single-positive, and TAg
single-positive (Fig. 1B). The
latter two groups were used as controls for the binary transgenic
system in response to tetracycline administration. In each group, one
animal received tetracycline treatment, the second received a placebo,
and the third received tetracycline first, followed by 7 days of
tetracycline withdrawal. The aim for the third group was to
evaluate the reversibility of transgene expression by tetracycline
regulation. To assess the tissue-specific regulation of the transgene
by tetracycline, we analyzed the livers and various other organs,
including the spleen. An identical scheme was followed for the
generation of tTA/lacZ mice. The expression of the
lacZ transgene was determined by
-galactosidase
activity.

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Fig. 1.
Construction of tTA transgenes and mouse
genotyping. A, schematic diagram of tTA constructs. Two
constructs were generated for the liver-specific tTA expression. The
first was constructed with the mouse albumin promoter upstream of tTA,
and the second was constructed with the mouse MUP promoter (see
"Materials and Methods"). SV40tIVSpA represents SV40
small t intron and polyadenylation sequences. B, mouse
genotyping. The genotypic pattern of Alb-TAg hybrid animals is
represented. The upper panel shows the results of Southern
blot analysis of genomic DNA digested with HindIII and
probed with the tTA cDNA; the isolated transgenic fragment was used
as a positive control. The same genomic DNA was subjected to PCR
analysis using TAg-specific primers, generating a 630-base pair
product, and the results are represented in the lower panel.
DNA isolated from COS-7 cells was used as a positive control. Based on
these findings, animals were classified as double-positive (positive
for both tTA and TAg), tTA single-positive, or TAg single-positive. Not
shown are the nontransgenic animals, which were negative in both
assays. kbp, kilobase pairs.
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Regulation of TAg Expression by Tetracycline--
After treatment,
the TAg mice described above were analyzed for TAg expression in the
liver, spleen, brain, tongue, kidney, lung, heart, ovary, skin, and
skeletal muscle. Fig. 2 shows the levels
of TAg expression in the livers and spleens of the Alb-TAg 3140 and
3123 mice and MUP-TAg 7552 mice. TAg expression was detected only in
the liver and not in the spleen. RT-PCR analysis for TAg mRNA
revealed a complete absence of TAg expression in all the organs listed
above, except liver (data not shown). In all these lines, TAg
expression was completely abrogated in the liver by tetracycline
treatment of the double-positive mice. In animals after 7 days of
tetracycline withdrawal (Fig. 2, third lane), TAg expression
resumed, although to a lower level compared with the placebo-treated
animals. This lower level may be attributed to a gradual disappearance
of the tetracycline from the body and/or a less-than-precise time
course of tetracycline release from the implanted pellet. The
single-positive transgenic mice (TAg or tTA) were completely negative
for TAg expression under all conditions, supporting the concept that
the binary components of this transgenic model are essential for
tetracycline-regulated gene expression. Finally, TAg expression could
be detected only in the liver and not in the other tissues, indicating
that the expression is tightly regulated only in the targeted tissue.
All these results suggest that a tightly regulated, liver-specific gene
expression by tetracycline administration can be achieved in this
transgenic model. However, it is important to mention that there were
considerable variations in the levels of TAg expression between the
different mouse lines. In MUP-TAg lines 7545 and 7016, none of the
double-positive animals expressed any detectable level of TAg (data not
shown).

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Fig. 2.
Regulation of TAg expression by tetracycline
in transgenic mice. Alb-TAg lines 3140 and 3123 and MUP-TAg lines
7552, 7545, and 7016 were generated as described under
"Results." The offspring were genotyped at 3 weeks of age,
and those harboring both tTA and TAg genes (Double) and
positive for only one of the transgenes (tTA or TAg) were used in the
study. Each group consisted of subgroups that were treated with either
tetracycline (Tetracycline +) or placebo pellets
(Tetracycline ). Some double-positive animals were first
treated with tetracycline, followed by 1 week of tetracycline
withdrawal (Tetracycline +(w)). Liver and spleen lysates
(100 µg of protein) were analyzed for TAg and actin protein content
by Western blot analysis using specific antibodies. The results are
representative of at least three independent experiments. Positive and
negative control lysates from cell lines (COS-7 and Huh-7 cells,
respectively) showed a barely detectable actin signal because the gels
were loaded with much lower protein content (5 µg). T antigen is a
85-kDa protein; actin is a 42-kDa protein.
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Regulation of lacZ Expression by Tetracycline--
To address
whether this tetracycline-regulated transgenic system can be used to
direct the conditional expression of any transgene, we evaluated the
expression of the lacZ reporter gene using the same
approach. Fig. 3 shows the levels of
lacZ expression in livers from these various
tTA/lacZ lines. The double-positive animals from
Alb-lacZ line 3140 and MUP-lacZ line 7552 demonstrated a tight regulation of lacZ expression in the
liver by tetracycline, similar to the results obtained with the TAg
lines above. Surprisingly, MUP-lacZ line 3123 did not show
any LacZ protein activity in the absence of tetracycline, which is
contrary to the results obtained with the corresponding TAg line. This
could be due to a relatively lower sensitivity of lacZ
detection in the livers of the animals. Similar to the tTA/TAg system,
gene expression was confined to the liver and was not detected in the
spleen. Discontinuation of tetracycline resulted in the resumption of
lacZ gene expression in the liver. These findings support
the notion that tetracycline-regulated gene expression can be
accomplished with any gene of interest. Table
I summarizes the findings of all TAg and
lacZ lines.

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Fig. 3.
Tetracycline-mediated regulation of
lacZ expression in the liver. The
Alb-lacZ and MUP-lacZ lines were generated by
breeding mice from the tetO-lacZ line with
different tTA lines and treated with tetracycline or a placebo, and the
liver lysates (100 µg of protein) were analyzed for -galactosidase
activity as described under "Materials and Methods." The
results are expressed in counts/min. Not shown are the LacZ protein
activities in the spleens of these mice and in the livers of
nontransgenic mice (negative controls), which were all within the
background level (<15,000 cpm). Tetracycline +(w),
tetracycline treatment, followed by 1 week of tetracycline
withdrawal.
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Table I
Regulated expression of TAg or lacZ in transgenic mouse livers
Alb-TAg or Alb-lacZ line 3140 was generated by crossing
Alb-tTA 3140 mice with tetO-TAg or
tetO-lacZ mice. Alb-TAg or Alb-lacZ
line 3123 was generated by crossing Alb-tTA 3123 mice with
tetop-TAg or tetop-lacZ mice. MUP-TAg lines 7552, 7545, and 7016 were generated similarly by crossing the respective
MUP-tTA mice with tetO-TAg or
tetO-lacZ mice. Genotypes of the offspring were
double-positive (bigenic for both tTA and TAg or lacZ;
Double), TAg single-positive (TAg or lacZ), or tTA
single-positive (tTA). Mice with each genotype were implanted with
either tetracycline (Tc) or placebo (P) pellets. A group of
double-positive animals was treated with tetracycline, killed, and
analyzed 1 week after dissipation of the tetracycline pellet (Tc/W).
Each group included at least three animals, and they all behaved
similarly. +, animals with TAg or lacZ expression; ,
animals that failed to express TAg or lacZ.
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Complete Suppression of Transgene Expression by
Tetracycline--
To determine whether there is a low level expression
of the transgene under suppressed conditions (by tetracycline) that is below the limit of the protein detection method above, total RNA was
isolated from all groups (double-positive and tTA and TAg single-positive mice) with or without tetracycline treatment and subjected to RT-PCR analysis. RT-PCR can detect as few as 1000 copies
of TAg transcripts, which translates to less than one copy/cell (10 µg of total RNA for RT-PCR, equivalent to ~104 cells),
and thereby is much more sensitive than Western blotting. The results
of the RT-PCR analysis of these RNA samples are shown in Fig.
4. Tetracycline treatment resulted in the
complete absence of TAg transcripts compared with the placebo group
(Fig. 4, lane 1). The tetracycline withdrawal group also
showed detectable levels of TAg RNA, supportive of the reversal of the
tetracycline regulation. Furthermore, the absence of TAg transcripts in
the TAg single-positive mice is consistent with the notion that this
transgene, in the absence of tTA, is completely silent.

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Fig. 4.
Complete suppression of TAg expression by
tetracycline in the livers of transgenic mice. Total cellular RNA
was isolated from liver and spleen tissues of various groups of mice
described under "Results" and from the positive control cell line
COS-7 (Positive RNA control). RNA was reverse-transcribed
(RT +) and PCR-amplified using specific primers and analyzed
on agarose gel. A positive control DNA was included in the assay
(Positive DNA control). As a control, reverse transcription
was also performed without reverse transcriptase (RT ).
Specificity of the amplified fragments was confirmed by Southern
blotting and probing with 32P-labeled TAg cDNA.
Tc Treatment +(w), tetracycline treatment, followed by 1 week of tetracycline withdrawal.
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Suppression of Hepatocellular Neoplasia by Tetracycline--
It is
well known that constitutive expression of TAg in transgenic mouse
models is oncogenic. At 4 weeks of age, double- and single-positive
animals from Alb-TAg line 3123 were subjected continuously to
tetracycline or placebo treatment for a period of 12 months and
killed. Gross morphological examination of the livers, spleens,
kidneys, lungs, hearts, brains, and skeletal muscles of all the animals
was performed. Evidence of hepatic neoplasia was present in three out
of five animals that were double-positive (Table
II). One of them had a large liver tumor.
Histological evaluation revealed hepatocellular adenoma with loss of
lobular architecture and compression of tissues adjacent to the adenoma (Fig. 5, F and G).
The hepatocytes within the adenoma were irregularly arranged with
considerable pleomorphism, large heterochromatic nuclei, many mitotic
figures, and intracellular fat vacuoles (lipidosis and steatosis). No
well defined portal triads or central veins or sinusoids were noticed.
The other two mice had multiple microscopic foci of nodular
hyperplasia, and one of them had a microadenoma. These hyperplastic
foci stood out as pale areas among the pink hepatic parenchyma and were
distributed in all lobes of the liver (Fig. 5, B and
C). Histological examination showed similar pathological changes to those of the adenoma described above. In contrast, none of
the tetracycline-treated animals developed any evidence of tumor in the
liver as revealed by histological examination (Fig. 5, A,
D, and E). In all cases, tumors were confined to
the liver only, which again supports the notion that this regulatable system can be precisely operated in a time-dependent and
tissue-specific manner. All other organs appeared grossly normal
without evidence of tumors. It is possible that they may have
microscopic pathology, but in the complete absence of TAg expression,
it is unlikely.
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Table II
Regulation of tumor induction by tetracycline treatment
Double- and single-positive animals derived from Alb-TAg line 3123 were
treated continuously with tetracycline or placebo for a period of 12 months and killed. Livers and other organs were examined for both gross
morphological changes and histological changes. All organs other than
the liver appeared normal in all mice. +/w, tetracycline treatment,
followed by 1 week of tetracycline withdrawal.
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Fig. 5.
Regulatable development of hepatic neoplasia
by tetracycline. Animals from Alb-TAg line 3123, which were
double- or single-positive for the transgenes, were treated with or
without tetracycline continuously for a period of 1 year. At the end of
the treatment, the animals were killed and examined for tumor
development in the liver and other organs as described under
"Results." Hematoxylin/eosin-stained liver sections were examined
for any histological changes. A shows the liver of a
tetracycline-treated double transgenic animal, which appeared
histologically normal and had no evidence of malignancy
(magnification × 2.5; the same view is shown in
D (magnification × 10) and E
(magnification × 40)). B and C show a low
power view (magnification × 2.5) of the livers from two
placebo-treated animals, in which a microadenoma (B,
arrow) and multiple hyperplastic foci (C,
arrowheads) can be observed. F and G
show the pathological features of the large hepatic adenoma from the
third animal (magnification × 10 and × 40, respectively).
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DISCUSSION |
In this study, we developed a binary transgenic system in which
the E. coli tetracycline regulon was genetically engineered to achieve a tightly regulated, liver-specific gene expression in
vivo. The liver-specific expression was accomplished using a
liver-specific promoter (the mouse albumin or mouse major urinary protein promoter) to drive the tetracycline-sensitive tTA in one transgenic lineage; the other transgenic lineage contains the reporter
gene (SV40 TAg or lacZ) under the control of the
tetO promoter, to which tTA, in the absence of tetracycline,
binds and activates. Analyses of the double transgenic mice showed that two of the three Alb-tTA lines and one of the two MUP-tTA lines exhibited the desirable phenotype of conditional expression in the
liver. The expression of both reporter transgenes could be completely
suppressed by tetracycline administration; such a suppression could be
reversed upon discontinuation of tetracycline.
The biological significance of this system was confirmed by the
phenotypic study of the TAg mice, in which the double transgenic animals developed liver neoplasia in the absence of tetracycline, and
the tetracycline-treated group had normal liver histology. SV40 T
antigen has been shown to be tumorigenic in transgenic mice (16-19).
Constitutive expression of TAg in the liver leads to massive
hepatomegaly and development of liver tumors by 5 months of age (20).
Although we have not performed a detailed kinetic study of tumor
development, three of five mice in the absence of continuous
tetracycline developed gross and histological hyperplasia and/or
neoplasia at 12 months of age. The less oncogenic potential of TAg in
our model probably reflects a lower level of TAg expression than in
other TAg transgenic mouse models. In a similar study by Ewald et
al. (21), the tetracycline-regulated expression of TAg was
targeted to the salivary glands of mice. These mice developed ductal
hyperplasia at 4 months of age. Importantly, tumor development could be
abolished by tetracycline administration at 4 months, but not at 7 months, of age. However, it is not clear whether the time or the stage
of the cancer is crucial for tumor suppression. In our model, we do not
know what the threshold time point is for tumor regression by
tetracycline. Further experiments are underway to address these issues.
Several transgenic mouse models have been developed using the
tetracycline-regulated system in various target tissues or organs. Gossen and Bujard (22) were able to achieve
tetracycline-regulated gene expression in lymphoid cells, and
Passman and Fishman (23) in cardiac muscles. In another study,
the rat liver enriched activator protein promoter was used for
liver-specific expression of tTA; however, reporter gene (luciferase)
expression could be detected in several organs such as spleen, heart,
brain, and liver under the induced conditions (24). In all these
studies, it is not clear whether one can achieve near-complete
suppression of the transgene by tetracycline administration with more
sensitive techniques such as PCR.
Other conditional expression systems have been applied to the
transgenic and knockout technology. The most notable one is the
cre-lox system (8, 25). cre is often
delivered by an adenoviral or retroviral vector to mice with the
lox-controlled transgene (26, 27). Although one can achieve
tissue-specific targeting using a tissue-specific promoter to express
cre, the recombination mediated by lox is often
incomplete, and the effect of viral vectors may have untoward
functional effects on the targeted tissues (28). Development of
cre transgenic mice under the control of tissue-specific
promoters can overcome the viral effects, but the advantage of specific
timing of conditional expression is lost. In addition, the
cre-lox-mediated recombination is not reversible, thereby mitigating the potential of an on-off regulation. Other conditional expression systems regulatable by pharmacologic agents such
as mifepristone (RU-486) (29), ecdysone (30), and rapamycin (FK506)
(31) have been developed recently. A system using the antiprogestin
mifepristone and progesterone receptor ligand-binding domain fusion
protein to regulate gene expression has been developed in transgenic
mice (32). Although several orders of magnitude of induction of the
transgene can be achieved in vivo, it is not clear whether
the dose of mifepristone used for induction exerts any unforeseen
biological effects on the animals or what the basal level expression of
the transgene is. Furthermore, the slow kinetics of regulation by
mifepristone in vivo limited the usefulness of this system
(32).
Our study has several biological implications. First, it is possible
keep the expression of a transgene completely silent in the liver and
to induce its expression at any desirable time point in a reversible
manner. This would allow a precise evaluation of the true biological
functions of any gene in the liver in the context of the defined
physiological or pathophysiological state of the animal without other
compensatory or epigenetic events. Second, this model system can be
applied to the silencing of an endogenous gene in a reversible,
time-dependent manner. For example, cre could be
placed under the control of the tetracycline-regulated system for
specific knockout of a gene, or an antisense transgene could be
designed to suppress the expression of an endogenous gene in the liver.
Third, by completely silencing the transgene during the fetal stage, it
is possible to avert the thymic selection process of a transgene as a
self-antigen and therefore to characterize the immunologic responses to
the transgene later in the animal's life. This can be applied to the
study of autoantigens or viral infections. The latter approach
is particularly valuable for hepatitis C, in which there is no
convenient small animal model. A previous study has reported the
induction of the hepatitis C virus core-specific immune response
using the cre-lox system to induce the expression of the core transgene in liver (28). However, the presence of a strong
anti-adenoviral immunity (delivery of cre by adenovirus to
the liver) precluded detailed characterization of the effects of an
anti-hepatitis C virus response in vivo. We are hopeful that
our model system will pave the way for the more precise study of any
endogenous or exogenous genes and their interactions with the immune
system in the context of viral immunopathology, hepatocarcinogenesis, autoimmunity, or other forms of liver disease.
 |
ACKNOWLEDGEMENTS |
We thank John Vergalla for excellent
technical assistance, Mark St. Clair for superb veterinary support, and
Shimon Efrat for providing the tetO-TAg mice. We thank
Donald J. Gardner for interpretation of the histology slides. We also
thank Andrew Leiter, the New England Medical Center/Tufts Center
for Gastroenterology Research on Absorptive and Secretory Processes
Digestive Disease Center, and the Massachusetts General Hospital Center
for Study of Inflammatory Bowel Disease for generating some of the
transgenic mice.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Liver Diseases
Section, DDB/NIDDK/NIH, Bldg. 10, Rm. 9B16, 10 Center Drive MSC 1800, Bethesda, MD 20892. Tel.: 301-402-1721; Fax: 301-402-0491; E-mail:
JakeL@bdg10.niddk.nih.gov.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009770200
 |
ABBREVIATIONS |
The abbreviations used are:
TAg, SV40 T antigen;
tTA, tetracycline-responsive transcriptional activator;
Alb, albumin;
MUP, major urinary protein;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-polymerase chain reaction.
 |
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