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INTRODUCTION |
Overexpression transgenic modeling is being used with increasing
frequency to investigate the processes involved in tissue homeostasis
and disease pathogenesis. This is nicely illustrated in the lung, where
the Clara cell 10-kDa protein (CC10)1 or
surfactant apoprotein-C promoters can be used to selectively target
genes of interest to the lung parenchyma and/or airway (1). In early
studies, these promoters were used to directly drive the expression of
transgenes in the lung. These studies provided impressive insights into
the chronic respiratory effector functions of inflammatory mediators
and the pathogenesis of asthma, adult respiratory distress syndrome,
lung development, and pulmonary fibrosis (2-9). In these systems, the
transgene is activated in utero and expressed in a
constitutive fashion thereafter. As a result, these modeling systems
are limited in their ability to accurately model waxing and waning
disease processes such as asthma and are unable to differentiate
transgene-induced phenotypic alterations that are due to alterations in
lung development from alterations that would otherwise be induced in an
adult/mature lung. These systems are also unable to appropriately study
genes whose products are toxic in early life and cannot be used to
define the natural history and/or reversibility of transgene-induced phenotypic alterations (1, 10, 11).
To deal with the limitations inherent in standard overexpression
modeling, a number of investigators have established transgenic systems
in which the expression of the transgene can be externally regulated.
Although a variety of approaches have been utilized, tetracycline-controlled expression systems have been employed most
frequently (10-17). Studies from our laboratory were the first to
establish the utility of this approach in the lung (10). This system is
based on the generation of transgenic mice with two molecular
constructs. In the first, the promoter of choice (CC10) drives the
expression of the reverse tetracycline transactivator (rtTA), a fusion
protein made up of the herpesvirus VP-16 transactivator and a mutant
Tet repressor from Escherichia coli. This
transactivating fusion protein requires doxycycline (dox) (a
tetracycyline derivative) for specific DNA binding. The second
construct contains multimers of the tetracycline operator
(tet-O), a minimal cytomegalovirus promoter, and the gene of
interest. In the absence of dox, the transactivator does not recognize
or weakly recognizes its specific target sequence (tet-O),
and target gene transcription occurs at low levels or not at all. The
addition of dox allows the transactivator to bind in trans to the
tet-O, activating the transgene of interest. This system has
been used successfully in our laboratory and others to define the
development-dependent and -independent processes that cause
alveolar enlargement in the lung, inflammatory events that may
contribute to the pathogenesis of pulmonary emphysema, cytokine-mediated protective events in oxygen toxicity, and crucial windows in lung development that define cytokine effector profiles (4,
10, 18-20). Detailed analyses of these and other rtTA-based systems
have, however, revealed some properties that restrict its application.
One limitation stems from the fact that rtTA can exhibit a degree of
residual affinity to tet-O in the absence of dox. This
manifests as definable levels of transgene activation and phenotype
induction in animals (or cells) that are not receiving dox (11, 16, 18,
21-25). Approaches that can be used to eliminate basal transgene leak
in vivo have not been well characterized.
The tet-controlled transcriptional silencer (tTS) is a
fusion protein made up of a mutant Tet repressor and the
KRAB-AB domain of the Kid-1 protein, a powerful transcriptional
repressor (24, 26). It binds to tet-O only in the absence of
dox. Therefore, in systems containing tTS and tet-O-driven
transgenes, tTS binds to the tet-O in the absence of dox and
inhibits the expression of the gene of interest. As dox is added to the
culture medium, the tTS dissociates from tet-O, relieving
the transcriptional suppression. At sufficient concentrations, the dox
also interacts with rtTA, allowing it to bind to tet-O and
activate the gene of interest. The tTS system has been shown to confer
exquisite regulating ability on transiently transfected and stably
transfected rtTA-regulated reporter genes in cells in culture (24, 25, 27). Surprisingly, the feasibility and efficacy of tTS in regulating basal gene expression in vivo in transgenic animals has not
been investigated.
We postulated that the principles involved in tTS inhibition of basal
gene expression in vitro are also applicable to the in
vivo state. To test this hypothesis, we generated transgenic mice
in which the CC10 promoter targeted tTS to the lung and bred these mice
with CC10-rtTA-IL-13 mice in which CC10 and rtTA were used to express
IL-13 in the lung/airway in an externally regulatable fashion. These
mice had been previously generated in our laboratory and shown to have
a quantifiable level of transgene leak in the absence of dox
administration (18). We then compared the IL-13 production and
phenotypes of the dual transgenic CC10-rtTA-IL-13 and the triple
transgenic CC10-rtTA/tTS-IL-13 mice. These studies demonstrate that tTS
totally abrogates basal transgene leak and phenotype induction in our
transgenic system. They also demonstrate that tTS mediates this
inhibition without decreasing the ability of dox to stimulate the
rtTA-regulated transgene or the ability of the transgene to induce a
tissue phenotype.
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EXPERIMENTAL PROCEDURES |
Generation of CC10-tTS-hGH Mice--
In order to express tTS in
a lung specific fashion, the construct CC10-tTS-hGH was generated (Fig.
1 A). The plasmid containing tTS, pTet-tTS, was obtained from Dr.
Andrew Farmer (CLONTECH Inc., Palo Alto, CA).
Oligonucleotide primers were synthesized that would introduce
HindIII and BamHI restriction enzyme sites 5' and
3' of the tTS coding region respectively. Furthermore a silent mutation
was introduced to disrupt the original BamHI site right before the stop codon. The primers were, tTSUP1: 5'-GTG AAC AAG CTT ATC
GCC TGG AGA CG-3' and tTSLO1: 5'-CTT AGT GGA TCC ATT TAC CAG GGG TCC
TCT CT TGC-3'. The tTS fragment was amplified by PCR and then inserted
between CC10 promoter and hGH polyadenylation and intronic sequence in
place of the rtTA sequences in construct CC10-rtTA-hGH that was
previously described by our laboratory (10). After verifying the
junction areas and tTS DNA by sequencing, the construct was isolated by
electrophoresis. The DNA was then purified through an Elutip-D column
following the manufacturer's instructions (Schleicher and Schuell,
Inc., Keene, NH) and dialyzed against microinjection buffer (25 mM Tris-HCl/0.5 mM EDTA, pH 7.5). Transgenic
mice were generated in (CBA X C57BL/6) F2 eggs using standard
pronuclear injection as previously described (6, 28).
The resulting animals and their progeny were genotyped using tail DNA
and Southern blot analysis with 32P-labeled tTS DNA as a
probe or PCR. For PCR reactions, primers were designed to cover the
area that was unique to the tTS transgene (Table
I). The primers were tTSUP2: 5'-GAG TTG
GCA GCA GTT TCT CC-3' and tTSL02: 5'-GAG CAC AGC CAC ATCTTC AA-3'. The
PCR protocol was 95 °C for 5 min; 30 cycles at 95 °C for 1 min;
60 °C for 1 min and 72 °C for 1 min and a final extension at
72 °C for 10 min. A product of 472 bp was expected and detected.
CC10-rtTA-IL-13 Mice--
CC10-rtTA-IL-13 transgenic mice were
generated as described by our laboratory (18). These dual transgenic
mice express IL-13 in the lung in an externally regulatable fashion.
The first construct in these mice, CC10-rtTA-hGH, contains the CC10
promoter, rtTA and human growth hormone (hGH) intronic and
polyadenylation sequences (hGH) (10). The second construct,
tet-O-IL-13-hGH, contains a tet-O and minimal
cytomegalovirus promoter, murine IL-13 cDNA and hGH intronic and
polyadenylation sequences. The constructs are illustrated in Fig. 1 A.
Generation of CC10-rtTA/tTS-IL-13 Triple Transgenic
Mice--
CC10-rtTA/tTS-IL-13 triple transgenic mice were generated by
breeding CC10-rtTA-IL-13 mice with CC10-tTS-hGH mice. The genotypes of
the progeny of these crosses were analyzed by PCR performed with tail
DNA with primers designed to determine if rtTA, tTS and/or IL-13 were
present (Table I). The protocols that were used to detect rtTA and
IL-13 have been previously described (2, 18). The PCR protocols for tTS
were noted above.
Dox Induction of IL-13--
All mice were maintained on normal
water until transgene activation was desired. At that time, dox (0.5 mg/ml) was added to the animal's drinking water. Sucrose (2%) was
also added to mask the bitter taste of dox and the dox water was kept
in dark brown bottles to prevent light-induced dox degradation.
Bronchoalveolar Lavage (BAL), Total Cell Counts and
Differentials--
Mice were euthanized and a median sternotomy was
performed. The trachea was then isolated via blunt dissection and small
caliber tubing was inserted and secured in the airway. Three successive volumes of 0.75 ml of PBS with 0.1% bovine serum albumin were then
instilled and gently aspirated. Total cell counts and differentials were evaluated as described previously (18, 21).
Cytokine Quantification--
BAL fluid aliquots were centrifuged
and the supernatants were stored at
70 °C until utilized. The
levels of IL-13 were determined immunologically using commercial ELISA
kits as per the manufacturer's instructions (R&D Systems, Minneapolis,
MN). Selected samples were concentrated 10 X by volume using Microcon
YM-10 following the manufacturer's protocol (Millipore Corporation,
Bedford, MA).
mRNA Quantification--
mRNA levels were assessed using
Northern analyses and/or RT-PCR as previously described by our
laboratory (18, 21). In the RT-PCR experiments, total RNA from mouse
lungs was prepared using Trizol reagent (Life Technologies, Inc., Grand
Island, NY) after treatment with DNase I following the manufacturer's
instructions. The RNA samples were reverse transcribed and
gene-specific primers were used to amplify selected regions of each
target moiety. Optimal annealing temperature and cyclings were derived
for each individual cytokine. Equal amounts of RNA were tested in each
reaction and
-actin was used as an internal standard. Amplified PCR
products were visualized using ethidium bromide gel electrophoresis and finally confirmed by nucleotide sequencing. IL-13, matrix
metalloproteinase (MMP)-12, eotaxin, monocyte chemotactic protein-1
(MCP-1), cathepsin K and
-actin mRNA were assessed using the
primers and conditions listed in Table I as described previously by our
laboratory (18, 21). In selected experiments the IL-13 RT-PCR product
was transferred to a nitrocellulose membrane and evaluated with a
labeled internal primer, 5'-TTT CCG CGG CTA CAG CTC CCT GGT TCT CTC-3'.
The tTS transcripts were evaluated as described above.
Histologic Evaluation--
In these experiments, mice were
euthanized, median sternotomies were performed and right heart
perfusion was accomplished with calcium and magnesium-free PBS to clear
the intravascular space. The heart and lungs were then removed en bloc,
inflated to 25 cm pressure with Streck tissue fixative solution (Streck Laboratories, Inc. Omaha, NE), embedded in paraffin and sectioned at 5 microns. Hematoxylin and eosin, Mallory's trichrome and periodic acid-Schiff with diastase (PAS) stains were performed in the Research Pathology Laboratory at Yale University.
Morphometric Analysis--
To evaluate the size of the alveoli,
lungs from transgenic and control mice were obtained, fixed to pressure
and sectioned and stained as described above. Alveolar chord length was
then measured as described previously by our laboratory (18).
Calculation of Histologic Mucus Index--
The histologic mucus
index is a measure of the number of mucus secreting airway epithelial
cells per unit basement membrane. It was calculated from PAS stained
histologic sections as previously described by our laboratory (18,
29).
Lung Volume and Compliance Assessment--
Lung volume and
compliance were assessed as described previously by our laboratory (18,
21). In these experiments mice were anesthetized, the trachea was
cannulated and the lungs were ventilated with 100% O2 via
a "T" piece attachment. The trachea was then clamped and oxygen
absorbed in the face of ongoing pulmonary perfusion. At the end of this
degassing, the lungs and heart were removed en bloc and
inflated with PBS at gradually increasing pressures from 0-30 cm. The
size of the lung at each 5 cm interval was evaluated via volume displacement.
Statistical Analysis--
Values are expressed as means ± S.E. As appropriate, groups were compared by analysis of variance with
Scheffe's procedure post hoc analysis, Student's t test or
nonparametric assessments (Wilcoxon's rank sum, Mann-Whitney
U test) using Stat View software for the Macintosh (Abacus
Concepts Inc., Berkeley, CA, USA).
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RESULTS |
Generation of CC10-tTS-hGH Transgenic Mice--
To characterize
the effects of tTS in our transgenic system, we generated multiple
lines of mice in which tTS was targeted to the lung using the CC10
promoter (Fig. 1A). The CC10-tTS-hGH construct was prepared, linearized and microinjected as previously described (6, 10). PCR using tail biopsy-derived DNA was used for
genotyping and RT-PCR of whole lung RNA was used to evaluate tTS gene
expression. Four transgene (+) founder animals were obtained from the
microinjection (Fig. 1B). Each was bred onto a C57BL/6 background and
independent lines were generated. In all cases, the transgene was
propagated in a Mendelian fashion. tTS mRNA was readily detected in
total lung RNA from transgene (+) animals. tTS mRNA was not
detected in lung RNA from transgene (-) littermate control animals. In
addition, tTS was not detected in a variety of extra thoracic organs in
transgene (+) or (-) progeny mice (Fig. 1C and data not shown). These
studies demonstrate that CC10 appropriately targets tTS to the murine
lung.

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Fig. 1.
Panel A: Schematic illustration of transgene
constructs used to generate CC10-tTS-hGH mice and CC10-rtTA/tTS-IL-13
triple transgenic mice. CC10-tTS-hGH construct was prepared and
microinjected as described under "Experimental Procedures." The
genotype and organ-specificity of tTS expression in CC10-tTS-hGH mice
were characterized. In panel B transgene (+) and transgene (-)
littermate control mice were identified by PCR with tail biopsy-derived
DNA and tTS primers. The results are compared with results obtained
with DNA from a negative control wild type mouse (negative) and
positive control CC10-tTS-hGH DNA (positive). In panel C RT-PCR was
used to define the organ specificity of transgene expression in
transgene (+) animals. The CC10-tTS-hGH mice were then crossbred with
CC10-rtTA-IL-13 double transgenic mice to generate CC10-rtTA/tTS-IL-13
triple transgenic mice.
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Characterization of CC10-tTS-hGH Mice--
To determine if tTS
altered lung structure, we compared the hematoxylin and eosin, PAS and
trichrome evaluations, alveolar morphometry and compliance of 1-3
month old CC10-tTS-hGH mice and wild type littermate controls. In all
cases, differences could not be appreciated (data not shown). Thus, tTS
expression did not alter the histology, morphometry or compliance of
the murine lung.
Effect of tTS on IL-13 Production in the Absence of Dox
Administration--
To determine if tTS altered the levels of IL-13
that were produced in the absence of dox administration, we compared
the levels of BAL IL-13 protein and lung IL-13 mRNA in double
transgenic (CC10-rtTA-IL-13) and triple transgenic
(CC10-rtTA/tTS-IL-13) mice. Single transgenic (CC10-tTS-hGH) and
transgene (-) littermate control animals were also evaluated. IL-13 was
not detected in the BAL fluid from the transgene (-) littermate
controls or the CC10-tTS-hGH animals. IL-13 (50-110 pg/ml) was,
however, readily appreciated in the BAL fluids from the dual transgene
(+) animals. In striking contrast to this finding, IL-13 protein was
undetectable in BAL fluids from uninduced triple transgenic animals
containing the tTS construct (Fig. 2).
After 10-fold BAL fluid concentration, IL-13 was still unable to be
detected in BAL fluids from triple transgenic mice, whereas significant
levels (0.9-1.5 ng/ml) of IL-13 were detected in similarly
concentrated BAL fluids from dual transgene (+) animals (Fig. 2). These
studies demonstrate that the tTS construct decreased the BAL IL-13
content of uninduced mice by at least 3 orders of magnitude. In accord
with these observations, mRNA encoding IL-13 was readily detected
via RT-PCR analysis in dual transgene (+) animals. In contrast, IL-13
mRNA was unable to be detected in lungs from triple transgenic tTS
mice even after 35 cycles of RT-PCR analysis (Fig. 2). When viewed in
combination, these studies demonstrate that the inclusion of the tTS
construct totally eliminated the background leak in our dual transgenic rtTA-regulated animals.

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Fig. 2.
Effects of tTS on IL-13 gene expression in
the absence of dox administration. Two month old wild type,
CC10-tTS-hGH (tTS +), CC10-rtTA-IL-13 (IL-13/rtTA +) and
CC10-rtTA/tTS-IL-13 (IL-13/rtTA +; tTS +) mice on normal water were
sacrificed, BAL was performed and whole lung RNA was isolated. The
levels of IL-13 in unconcentrated (A) and 10 X concentrated (B) BAL
fluids were evaluated by ELISA. Each value represents the mean ± S.E. of a minimum of 4 mice. In all cases the levels of IL-13 in BAL
fluids from CC10-rtTA/tTS-IL-13 mice were similar to those in the BAL
fluids from wild type mice and at or below the limits of detection of
the assay (~10 pg/ml). In panel C RT-PCR was used to characterize the
levels of IL-13 mRNA in RNA from lungs of these mice.
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Effects of tTS on Phenotype in the Absence of Dox
Administration--
Lungs from CC10-rtTA-IL-13 animals that did not
receive dox were enlarged and had larger alveoli and enhanced
compliance when compared with lungs from transgene (-) littermate
controls (Fig. 3). They also manifest
increased BAL cellularity, eosinophil, lymphocyte and macrophage rich
BAL and tissue inflammation, mucus metaplasia and enhanced accumulation
of eotaxin, MCP-1, MMP-12 and cathepsin K mRNA (Fig.
4, Table II
and data not shown) (2, 18). In contrast to these findings, the lung
volumes, alveolar size and pulmonary compliance of triple transgenic
tTS containing animals were unable to be differentiated from the same
parameters in transgene (-) littermate control animals or CC10-tTS-hGH
mice (p < 0.01 comparing triple and dual transgene (+)
animals for each parameter) (Fig. 3). In addition, the BAL
abnormalities, mucus metaplasia and the eotaxin, MCP-1, MMP-12 and
cathepsin K gene expression that were prominent features of the dual
transgene (+) animals were totally abrogated in the triple transgene
(+), tTS-containing animals (Figs. 4 and Table II). These studies
demonstrate that the inclusion of the tTS construct totally abrogated
the inflammatory, structural, mucus, physiologic and target gene
alterations induced by the basal IL-13 leak in this murine transgenic
system.

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Fig. 3.
Effect of tTS on pulmonary phenotype in the
absence of dox administration. Two month old wild type,
CC10-tTS-hGH (tTS +), CC10-rtTA-IL-13 (IL-13/rtTA +) and
CC10-rtTA/tTS-IL-13 (IL-13/rtTA +; tTS +) mice on normal water were
sacrificed and their phenotypes were compared as described under
"Experimental Procedures." Lung volumes, alveolar chord length, and
pulmonary compliance are illustrated in Panels A, B and C respectively.
(*p < 0.01 compared with CC10-rtTA-IL-13
animals).
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Fig. 4.
Effect of tTS on mucus and target gene
responses in the absence of dox administration. Two month old wild
type, CC10-tTS-hGH (tTS +), CC10-rtTA-IL-13 (IL-13/rtTA +) and
CC10-rtTA/tTS-IL-13 (IL-13/rtTA +; tTS +) mice on normal water were
sacrificed and their phenotypes were compared as described under
"Experimental Procedures." Panel A illustrates the airway mucus
metaplasia detected on PAS stains of lung tissues from these animals.
In Panel B, RT-PCR was used to compare the levels of mRNA encoding
eotaxin, MCP-1, MMP-12 and cathepsin K in the presence and absence of
tTS.
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Table II
Effect of tTS on BAL cell recovery
One-month-old wild type, CC10-tTS-hGH (tTS +), CC10-rtTA-IL-13
(IL-13/rtTA +), and CC10-rtTA/tTS-IL-13 (IL-13/rtTA +; tTS +) mice were
placed on normal water (Dox ) or Dox water (Dox +). When they were 2 months of age they were sacrificed, and BAL cell recovery was
quantitated.
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Effects of tTS on Dox-Induction of IL-13 and the Generation of the
IL-13 Phenotype--
For the CC10-tTS-hGH construct to have maximal
utility, it needs to suppress transgene expression in the absence of
dox without suppressing the induction of the transgene after dox
administration. To determine if tTS had effects on dox induction, we
compared the IL-13 elaboration and phenotype induction in dox-treated
dual transgene (+) and triple transgene (+) animals. As previously reported, dox was a potent inducer of IL-13 protein production and
mRNA accumulation in these dual transgenic animals (18). This
induction was noted within 48 h and persisted with chronic dox
administration. This inducibility was not significantly altered by tTS
since virtually identical levels of BAL IL-13 protein were seen in
comparably dox treated dual and triple transgenic animals (Fig.
5). In addition, comparable levels of
IL-13 mRNA were also noted in these dox-treated dual and triple
transgenic animals when assessed via RT-PCR or Northern analysis (Fig.
5 and data not shown). Thus, the presence of the tTS construct did not
significantly diminish transgene inducibility in our transgenic
system.

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Fig. 5.
Effect of tTS on dox induction of IL-13 gene
expression. One month old wild type, CC10-tTS-hGH (tTS +),
CC10-rtTA-IL-13 (IL-13/rtTA +) and CC10-rtTA/tTS-IL-13 (IL-13/rtTA +;
tTS +) mice were placed on dox water for 1 month and sacrificed. BAL
IL-13 levels were determined by ELISA (Panel A). RT-PCR was used to
evaluate the levels of IL-13 mRNA in lungs from these animals
(Panel B). (* p < 0.05 compared with wild type and
CC10-tTS-hGH mice.)
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As noted above, modest increases in lung and alveolar volume, enhanced
lung compliance, an eosinophil, lymphocyte and macrophage rich
inflammatory response, mucus metaplasia and eotaxin, MCP-1, MMP-12 and
cathepsin K gene expression were seen in dual transgenic mice on normal
water. Administration of dox caused a further increase in IL-13
production and an increase in the intensity of all of these phenotypic
features (Figs. 6 and
7, Table II and data not shown). In the
triple transgenic mice, no phenotype was seen in the absence of dox
administration. After dox administration, however, enhanced lung
volumes, alveolar enlargement, enhanced pulmonary compliance, increased
BAL cellularity, eosinophil, lymphocyte and macrophage rich tissue and
BAL inflammation and mucus metaplasia were all readily appreciated. In
addition, RT-PCR and Northern analysis demonstrated prominent eotaxin,
MCP-1, MMP-12 and cathepsin K gene expression. In all cases, the
magnitude of the alterations in each of these parameters in the
dox-treated triple transgenic mice were comparable to those in
dox-treated dual transgenic animals (Figs. 6 and 7, Table II and data
not shown). Thus, the tTS construct, while inhibiting basal IL-13
production, did not diminish the ability of dox to induce IL-13
elaboration or the ability of IL-13 to induce its tissue phenotype.

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Fig. 6.
Effect of tTS on the dox-induced IL-13
phenotype. One month old wild type, CC10-tTS-hGH (tTS +),
CC10-rtTA-IL-13 (IL-13/rtTA +) and CC10-rtTA/tTS-IL-13 (IL-13/rtTA +;
tTS +) mice were placed on dox for one month and sacrificed. Lung
volumes, alveolar chord length, and pulmonary compliance are
illustrated in Panels A, B, and C, respectively. (*p < 0.05 compared with wild type and CC10-tTS-hGH animals only.)
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Fig. 7.
Effect of tTS on dox-induced mucus and target
gene responses. One month old wild type, CC10-tTS-hGH (tTS +),
CC10-rtTA-IL-13 (IL-13/rtTA +) and CC10-rtTA/tTS-IL-13 (IL-13/rtTA +;
tTS +) mice were placed on dox for one month and sacrificed. Panel A
illustrates the airway mucus metaplasia detected on PAS stains of lung
tissues from these animals. In Panel B, RT-PCR was used to compare the
levels of mRNA encoding eotaxin, MCP-1, MMP-12 and cathepsin K in
the presence and absence of tTS.
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DISCUSSION |
Tetracycline responsive regulatory systems have been shown to
control gene expression in cultured cells and whole organisms including
yeast, Drosophilia, plants, mice and rats (11). A major
requirement for these systems is a target transgene that is under tight
"outside" control. Specifically, these systems should have
negligible levels of transgene expression in the absence of
tetracycline analogue administration and high levels of transgene expression after tetracycline/dox induction. Unfortunately, these goals
are not always achievable. Depending on the experimental conditions
that are employed and the site(s) of integration of the transgenic
constructs, enhancer sequences near the target gene have been
repeatedly demonstrated to increase basal target gene expression
thereby compromising the tight regulation that is desired (11, 16, 18,
21-25). In some circumstances, the leak that results is acceptable and
does not negate the ability of the experimental system to appropriately
address the hypothesis that is being investigated (10, 11, 18). In
others, however, the leak is more problematic. The confounding effects
of transgene leak can be easily appreciated in the context of the
CC10-rtTA-IL-13 mice generated in our laboratory. When these mice were
initially generated on a mixed CBA/C57BL/6 genetic background, very low levels of BAL IL-13 and marginally detectable phenotypes were appreciated (18). However, as breeding on to pure murine genetic backgrounds was accomplished, basal levels of IL-13 increased and a
more impressive phenotype was appreciated. Since the tTS has been shown
to eliminate/control basal leak in in vitro cell culture
systems (11, 24, 25), we hypothesized that this approach could also be
used to control basal transgene leak in in vivo transgenic
systems. To test this hypothesis, we generated mice in which tTS was
targeted to the lung, bred these mice with a dual transgenic
CC10-rtTA-IL-13 mice and then compared the IL-13 production and the
phenotypes of the resulting dual and triple transgenic mice before and
after dox administration. These studies demonstrate that the
incorporation of tTS in our transgenic system decreased basal transgene
leak to undetectable levels and totally eliminated the IL-13-induced
phenotype in the absence of dox induction. These studies also
demonstrate that tTS did not alter the ability of dox to increase IL-13
elaboration and induce a full-blown IL-13 phenotype. When viewed in
combination, these studies demonstrate that tTS converted our
rtTA-based system from one with low and high levels of transgene
expression to one that now has true off and on settings in the absence
and presence of dox induction.
When transgenes are inserted into cells, or whole organisms like
transgenic mice, the levels of basal transgene expression are regulated
by a number of factors including the site of integration in the host
genome and the number of copies of the integrated transgene. When
rtTA-based regulatory systems are employed an ideal integration site
would be one that minimizes cross talk between the minimal promoter in
the target construct and nearby cis-acting enhancers while maintaining
dox inducibility. In theory, this type of integration event can be
obtained if large numbers of cellular transfections (for in
vitro studies) or transgenic microinjections (for in
vivo studies) are undertaken (11, 24). This can, however, be a
very large and, at times, unachievable undertaking. The alternative
approach is to increase the yield of functional dual transgenic
offspring using transfection or microinjection approaches in which both
transgenic constructs are transferred simultaneously (see below) and
concurrently insert constructs that shield the
tet-controlled transgenic unit from extraneous activation.
We chose to do this by simultaneously microinjecting the two transgenic
constructs in our transgenic system and then adding tTS, a fusion
protein made up of the Tet-R and a transcriptional silencing domain.
This protein binds tet-O in the absence of dox, protecting
it from outside activation. Importantly, it also releases in the
presence of dox allowing rtTA to bind to tet-O and induce transgene activation. The Tet-R-based rtTA and tTS regulatory proteins
both bind tet-O as homodimers. In theory, when they are co-expressed they can also form tTS/rtTA heterodimers that do not
function appropriately and compromise the overall inducibility of this
system (24, 30). To ensure that this did not occur, the tTS construct
has been modified at its dimerization surface to prevent this rtTA
interaction. Our studies show that tTS provides a powerful shield for
and tightens the regulation of tet-O constructs that are not
integrated in an "ideal" location. They also demonstrate that tTS
does not alter dox inducibility, suggesting that the modification of
the dimerization surface successfully prevented the formation of large
numbers of heterodimeric molecules. It is important to point out that,
in addition to the tTS, other approaches have been described that have
the ability to control basal transgene leak. They include the use of
insulators such as those characterized in Drosophilia
chromatin (31) and novel new rtTA mutants that expand the range and
sensitivity of rtTA systems (22). In contrast to tTS, the utility of
each of these approaches in transgenic mice has not been established.
If subsequent studies demonstrate that these approaches work in
transgenic systems, it will be important to undertake studies that
compare and contrast these and the tTS systems to define the relative
merits and limitations of each.
Tight control of tet-regulated transgenes requires
appropriate shielding of the minimal promoter in the expression
construct. As a result, it has been proposed that optimal control can
not be achieved when both transgenic constructs are transferred
simultaneously either within a single construct or by co-injection of
the two plasmids in the rtTA system (11). This has, however, never been formally proven. In addition, there are a number of reasons why it is
often impractical to generate dual transgenic mice by breeding single
transgene (+) animals. First, there are fewer dual transgene (+)
animals per litter using this approach. This increases the breeding
time required for the experiment and the effort required for the
genotyping of progeny mice. In addition, dual transgene (+) animals
that were generated by breeding of single transgene (+) animals do not
lend themselves readily to experiments in which these animals are bred
to mice with targeted null mutations. This makes it difficult for
investigator to define the contributions to the transgene-induced
phenotype of genes that are regulated by the overexpressed transgene.
It is well documented in studies from our laboratory and others that
dual transgene-rtTA-based systems with acceptable levels of leak can be
achieved using an approach in which both constructs are microinjected
simultaneously (10, 11, 18, 21). The present studies also demonstrate that the tTS optimizes the regulation of these transgenes when significant basal leak is noted. Thus, it is reasonable to hypothesize that, in the future, inducible transgenic systems may be able to be
generated most efficiently using an approach in which the tTS
construct, rtTA construct and tet-O-transgene construct are all simultaneously microinjected or included in a single construct. Since transgenes tend to insert into the genome in a head to tail fashion, this would provide transgene (+) animals with tight transgene control in which all 3 constructs are passed to progeny as if they were
a single gene. This would minimize the breeding and genotyping required
for phenotypic characterization. It would also facilitate experiments
in which mice with null mutations of downstream genes are used to
define the role(s) that these genes play in mediating the
transgene-induced phenotype.
IL-13 is a 12 kDa cytokine produced in large quantities by T helper
(Th) 2 and lesser quantities by Th1 cells (32, 33). Studies from our
laboratory and others have demonstrated that IL-13 plays a central role
in the pathogenesis of airways disorders such as asthma and chronic
obstructive pulmonary disease (COPD) and can confer protection in the
setting of oxidant-induced lung injury (2, 18, 20, 34, 35). This is
nicely illustrated in the CC10-rtTA-IL-13 animals in which
dox-induction resulted in a phenotype that includes mucus metaplasia,
eosinophil and lymphocyte-rich inflammation, lung and alveolar
enlargement and an enhanced ability to tolerate the toxic effects of
100% O2 (18, 20). Since dox-induction of IL-13 caused an
increase in alveolar size in adult mice, the CC10-rtTA system allowed
us to demonstrate that IL-13 did not cause alveolar enlargement by
blocking alveolar development. It also allowed us to define the roles
of matrix metalloproteinases (MMPs) and cathepsins in the generation of this response and the mechanisms of IL-13-induced protection in hyperoxic acute lung injury (18, 20). Our knowledge of the pathogenesis
of asthma, COPD, acute lung injury and virtually all other disorders is
strikingly limited as regards the reversibility of the injuries
involved in these disorders and the nature of the repair responses that
they cause. This deficiency is due, in part, to a lack of modeling
systems that allow defined injuries to be applied and then fully
removed in an appropriately controlled fashion. The basal leak in the
dual transgenic, CC10-rtTA system renders it similarly lacking in this
regard. The triple transgenic CC10-rtTA-tTS system, however, can be
used in a true "on/off" fashion. It is thus reasonable to believe
that this system can be used to define the natural history of in
vivo injury and repair responses with a level of precision that
has not been previously achievable.
In summary, these studies demonstrate that the incorporation of tTS
into rtTA-based externally regulatable overexpression transgenic
systems optimizes the regulation of transgene expression in
vivo. By eliminating basal transgene leak and phenotype induction without altering the dox-inducibility of rtTA-regulated transgenes, tTS
converts the rtTA-based system from one with low and high levels of
gene expression to one with true "off" and "on" regulation. The
"off/on" nature of this regulation will be very useful in studies
in which toxic genes are expressed in a temporally restricted fashion
and studies in which critical windows of development and the natural
history of injury and repair are being precisely defined. The tight
control that is seen with the combined use of tTS and rtTA should now
be the standard against which other inducible overexpression systems
are judged.