Conditional Disruption of the Aryl Hydrocarbon Receptor Nuclear Translocator (Arnt) Gene Leads to Loss of Target Gene Induction by the Aryl Hydrocarbon Receptor and Hypoxia-Inducible Factor 1
Shuhei Tomita1,
Christopher J. Sinal1,
Sun Hee Yim and
Frank J. Gonzalez
Laboratory of Metabolism National Cancer Institute
Bethesda, Maryland 20892
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ABSTRACT
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To determine the function of the aryl hydrocarbon
receptor nuclear translocator (ARNT), a conditional gene knockout mouse
was made using the Cre-loxP system. Exon 6, encoding the conserved
basic-helix-loop-helix domain of the protein, was flanked by loxP
sites and introduced into the Arnt gene by standard gene
disruption techniques using embryonic stem cells. Mice homozygous for
the floxed allele were viable and had no readily observable phenotype.
The Mx1-Cre transgene, in which Cre is under control of the
interferon-
promoter, was introduced into the
Arnt-floxed mouse line. Treatment with
polyinosinic-polycytidylic acid to induce expression of Cre resulted in
complete disruption of the Arnt gene and loss of ARNT
messenger RNA (mRNA) expression in liver. To determine the role of ARNT
in gene control in the intact animal mouse liver, expression of target
genes under control of an ARNT dimerization partner, the aryl
hydrocarbon receptor (AHR), was monitored. Induction of CYP1A1, CYP1A2,
and UGT1*06 mRNAs by the AHR ligand
2,3,7,8-tetrachlorodibenzo-pdioxin was absent in
livers of Arnt-floxed/Mx1-Cre mice treated with
polyinosinic-polycytidylic. These data demonstrate that ARNT is
required for AHR function in the intact animal. Partial deletion of the
Arnt allele was found in kidney, heart, intestine, and
lung. Despite more than 80% loss of the ARNT expression in lung,
maximal induction of CYP1A1 was found, indicating that the expression
level of ARNT is not limiting to AHR signaling. Cobalt chloride
induction of the glucose transporter-1 and heme oxygenase-1 mRNAs was
also markedly abrogated in mice lacking ARNT expression, suggesting an
inhibition of HIF-1
activity. These studies establish a critical
role for ARNT in AHR and HIF-1
signal transduction in the intact
mouse.
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INTRODUCTION
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ARNT is a member of the basic helix-loop-helix, Per/AHR/ARNT/Sim
(bHLH-PAS) family of transcription factors and serves as a dimerization
partner for a number of the other family members. Among the partners
for ARNT are the aryl hydrocarbon receptor (AHR) (1, 2);
hypoxia-inducible factor (HIF-1
) (3); Sim, a transcription factor
involved in central nervous system development (4); and Per, a protein
involved in regulating the circadian clock (5, 6). The nuclear receptor
counterpart of ARNT is the RXR family that consists of three members
that partner with a number of other nuclear receptors to form
transcriptionally active complexes (7). ARNT can also function as a
homodimer to activate transcription from E box elements, albeit the
physiological significance of this activity is unknown (8). Based upon
in vitro trans-activation studies showing that it
is a promiscuous dimerization partner in the bHLH-PAS superfamily, ARNT
would be expected to have important roles in mammalian development and
physiological homeostasis. This is reflected in part by results
obtained with the ARNT-null mouse that is embryonic lethal due to a
defect in angiogenesis and placental development (9, 10).
To explore the function of ARNT in an intact animal model, the Cre-loxP
conditional knockout strategy was used to disrupt the gene in a
tissue-selective manner in adult mice (11, 12). The production of an
Arnt-floxed allele and the consequence of successful
disruption of the gene in liver using the Mx1-Cre transgene is
described. These studies establish with certainty that ARNT is required
for AHR and HIF-1
signal transduction in the intact animal.
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RESULTS
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Preparation of an ARNT Conditional-Null Mouse
An oligonucleotide probe against the mouse Arnt gene
bHLH region was used to isolate a genomic clone. The phosphoglycerate
kinase (PGK)-neo cassette, flanked by loxP sites, was inserted
into intron 5, and a second loxP site was placed in intron 6 (Fig. 1
). Cre-mediated recombination would
result in deletion of exon 6 that encodes the bHLH region of the
transcription factor protein. The targeting vector also contained a
PGK-thymidine kinase (PGK-TK) for use as a negative selection marker in
embryonic stem (ES) cells using ganciclovir.

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Figure 1. Targeted Modification of the Arnt
Gene
The Arnt gene, targeting construct, targeted allele, and
schema of Cre-mediated deletion of the Arnt gene are
shown. Hatched triangles represent loxP sites, and the
small arrow above the PGK-neo cassette shows the
direction of transcription that is opposite the direction of
transcription of the ARNT gene. The BamHI restriction
fragments monitored and detected with the 200-bp probe located in
intron 4 are denoted by double end arrows. The
restriction sites are abbreviated as follows: A, AvrII;
B, BamHI; E, EcoRI; S,
SacI; X, XhoI.
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Transmission of the Arnt-floxed allele could be monitored
using a single set of PCR primers designed from sequence flanking the
floxed site located in intron 6 (Fig. 2
).
The Mx1-Cre transgene could also be monitored by PCR. Mice homozygous
for the Arnt-floxed allele were viable and had no observable
phenotype, indicating that the modified Arnt allele was
functional; a disrupted Arnt gene would have resulted in
early embryonic lethality (9, 10). This indicates that the presence of
the PGK-neo cassette in intron 5 did not disrupt gene function. The Cre
recombinase was introduced into the Arnt-floxed mouse by
crossing with mice expressing the Mx1-Cre transgene (11). This
transgene is under control of the interferon-
promoter and results
in near-complete loxP site-mediated recombination in liver and
macrophages upon treatment of mice with the interferon inducer
polyinosinic-polycytidylic (pIpC) (11, 13, 14).

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Figure 2. Detection of Germline Transmission of the
Arnt-Floxed Gene
A, PCR-based genotyping strategy used for detection of the
loxP-targeted Arnt-floxed allele and the Mx1-Cre
transgene. Triangles and arrows represent
the loxP sites and PCR primers used, respectively. The restriction
sites are as follows: A, AvrII and E,
EcoRI. B, Representative PCR genotyping result for the
Arnt-targeted allele and Cre recombinase transgene
obtained using mouse tail genomic DNA. PCR products were separated on a
2.5% agarose gel and visualized by ethidium bromide staining. A 100-bp
ladder marker was used as a size marker.
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In Arnt-floxed mice treated with pIpC, the
Arnt exon 6 was deleted more than 95% in liver and thymus
(Fig. 3
). Partial deletion was found in
kidney, heart, lung, and small intestine, consistent with earlier
results using the Mx1-Cre transgene on other floxed genes, where
partial recombination was found (11, 13, 14). The deleted allele was
also observed in thymus of untreated Arnt-floxed/Mx1-Cre
mice, indicating the possibility of leaky expression of the Cre
transgene as a result of activation of the Mx1-Cre promoter by
endogenous interferon. In addition, a fragment corresponding to
deletion of the PGK-neo was detected in thymus DNA of untreated
Arnt-floxed/Mx1-Cre mice; this band was not seen in any of
the other tissue DNA samples from either untreated or pIpC-treated
Arnt-floxed/Mx1-Cre mice. Deletion of Arnt exon 6
was complete and unchanged in liver at 3 days and 21 days after pIpC
administration, indicating that liver stem cells with no disrupted
allele did not repopulate the liver to any appreciable degree (data not
shown). During this period, the mice exhibited no observable
phenotype.

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Figure 3. Detection of the Extent of Cre-Mediated
Recombination in Different Tissues of pIpC-Treated
Arnt-Floxed/Mx1-Cre Mice
Arnt-floxed and Arnt-floxed/Mx1-Cre
mice were treated with saline or pIpC, and tissues were harvested 3
days later. DNA (10 µg) from each tissue was digested with
BamHI and subjected to electrophoresis on a 0.8%
agarose gel, transferred to a nylon membrane, and hybridized with the
32P-labeled SalI to XbaI
fragment shown in Fig. 1 . The estimated sizes of the fragments
containing the floxed allele, deleted exon 6-deleted allele, and allele
lacking the PGK-neo cassette are 8.0, 6.5, and 4.5 kb, respectively.
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Loss of AHR Function in ARNT Conditional- Null Mice
To confirm that deletion of the bHLH-encoding exon resulted
in loss of ARNT gene expression, Northern blotting was carried out. In
Arnt-floxed/Mx1-Cre mice treated with pIpC, ARNT messenger
RNA (mRNA) was not detected in liver tissue compared with that in
vehicle-treated controls (Fig. 4
). Mice
lacking the Mx1-Cre transgene, but homozygous for the
Arnt-floxed allele, had normal levels of ARNT mRNA after
treatment with pIpC (data not shown). These data indicate that deletion
of the exon 6, mediated by Cre recombinase under control of the
interferon-
promoter, results in the production of an aberrant ARNT
mRNA that is not stable. ARNT expression was lower, but not eliminated,
in other tissues in which only partial recombination was found. In
kidney, heart, and lung, about 3050% of ARNT mRNA remained after
pIpC treatment. Expression of ARNT mRNA in intestine was not
significantly lower despite about 30% deletion in this tissue.

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Figure 4. Expression of ARNT mRNA in Different Tissues of
Arnt-Floxed/Mx1-Cre Mice
Total RNA (10 µg) was prepared from the indicated tissues of mice
treated with pIpC or saline and was subjected to Northern blot
analysis. A 32P-labeled cDNA probe was used to detect ARNT
mRNA. ß-Actin was used as a loading control.
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To determine the functional consequences of loss of ARNT expression,
target genes for the AHR were examined in mice lacking hepatic ARNT. To
assess whether gene induction mediated by AHR was reduced or eliminated
by disruption of expression of ARNT, Arnt-floxed/Mx1-Cre
mice treated with pIpC were administered the potent inducer
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mice lacking
ARNT expression, induction of the Cyp1a1 and
Cyp1a2 genes was abolished in liver (Fig. 5
). As previously demonstrated (15),
induction of these genes by TCDD was robust in wild-type mice,
Arnt-floxed/Mx1-Cre mice pretreated with vehicle instead of
pIpC, and Arnt-floxed mice without Cre and pretreated with
pIpC. Similar results were obtained with a second AHR target gene,
Ugt1t106 (Fig. 6
). It is
noteworthy that constitutive expression of CYP1A2 mRNA was lower when
ARNT expression was disrupted. TCDD administration partially restored
expression of the CYP1A2 mRNA.

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Figure 5. Expression of Hepatic CYP1A1 and CYP1A2 mRNA in
Arnt-Floxed/Mx1-Cre Mice
The mice were injected with pIpC or saline followed by administration
of TCDD or corn oil. Arnt-floxed mice lacking the Cre
transgene were used as a control for the effects of pIpC alone on basal
and induced expression. Total liver RNA (10 µg) was subjected to
Northern blot analysis using 32P-labeled cDNA probes
against mouse CYP1A1 and CYP1A2 mRNAs. ß-Actin was used as a mRNA
loading control.
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Figure 6. Expression of Hepatic UGT1*06 mRNA in
Arnt-Floxed/Mx1-Cre Mice
Arnt-floxed/Mx1-Cre and Arnt-floxed
mice were treated with pIpC followed by treatment with TCDD or corn
oil. Total liver RNA (10 µg) was subjected to Northern blot analysis
using 32P-labeled cDNA probes against mouse UGT1*06 mRNA.
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TCDD induction of CYP1A1 was also monitored in other nonhepatic
tissues. Among the tissues examined, partial loss of CYP1A1 induction
was only found in the heart of Arnt-floxed/Mx1-Cre mice
treated with TCDD (Fig. 7
). The extent of
induction by TCDD was not changed in kidney, intestine, or lung.

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Figure 7. CYP1A1 mRNA Induction in Extrahepatic Tissues of
Arnt-floxed/Mx1-Cre Mice
Animals were treated with pIpC or saline, followed by treatment with
TCDD or corn oil. Arnt-floxed mice lacking the Cre
transgene were used as a control for the effects of pIpC alone on basal
and TCDD-induced CYP1A1 expression. Total RNA (10 µg) was prepared
from the indicated tissues and subjected to Northern blot analysis
using a 32P-labeled mouse CYP1A1 cDNA probe. ß-Actin was
used as a loading control.
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Loss of HIF-1
Function in ARNT Conditional- Null Mice
To further examine the functional consequences resulting
from loss of ARNT expression, target genes for HIF-1
were assessed
in mice lacking hepatic ARNT. The basal steady state levels of the
mRNAs for aldolase A, glucose transporter-1 (Glut-1), and heme
oxygenase-1 were not affected by deletion of ARNT after pIpC treatment
(Fig. 8
). Treatment of the mice with
CoCl2, a chemical known to mimic physiological
hypoxia in animals (16) and cell culture (17), dramatically increased
the expression of these genes in the livers of mice with functional
Arnt expression. On the other hand, in mice lacking
Arnt expression, variable levels of abrogation of the
inducible expression of these HIF-1
-regulated genes was observed.
For example, hypoxic induction of hepatic heme oxygenase-1 was markedly
reduced and that of Glut-1 was lost entirely in mice treated with
CoCl2 and lacking ARNT (Fig. 8
). In contrast,
only a modest loss of aldolase A mRNA induction occurred as a result of
Arnt deletion. Analysis of HIF-1
mRNA levels indicated
that inhibition of the hypoxic response could not be attributed to
alterations in the expression of this ARNT dimerization partner and
that ARNT has no influence on the expression of HIF-1
. Treatment of
mice lacking the Mx1-Cre transgene with pIpC had little or no effect on
the expression of any of the genes examined in this experiment.

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Figure 8. Expression of HIF-1 mRNA and mRNA Derived from
Putative HIF-1 Target Genes in Arnt-Floxed/Mx1-Cre
Mice
The mice were injected with pIpC or saline, followed by administration
of CoCl2 or saline vehicle. Arnt-floxed mice
lacking the Cre transgene were used as a control for the effects of
pIpC alone on basal and induced expression. Total liver RNA (10 µg)
was subjected to Northern blot analysis using the
32P-labeled cDNA probes indicated in the figure. ß-Actin
was used as a mRNA loading control.
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DISCUSSION
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Standard gene targeting of Arnt in ES cells results in
embryonic lethality that is thought to be due to disruption of the
HIF-1
-dependent angiogenesis and vascularization required for
placental and embryo development (9, 10). To circumvent this problem,
the Cre-loxP conditional gene disruption system was used to study the
function of the ARNT protein in the intact mouse. By use of this
system, a gene can be inactivated in specific tissues at any age,
allowing for the first time examination of the functional effects of
ARNT deletion in the intact mouse.
Mice with a floxed Arnt gene were found to be viable,
with no observable phenotype, indicating that insertion of the
selectable marker PGK-neo into intron 5 in the opposite transcriptional
orientation from the Arnt gene did not interfere with
Arnt transcription and pre-mRNA splicing. The Mx1-Cre
transgene, in which the Mx1 interferon-
promoter controls Cre
expression, was used to disrupt the Arnt gene (11).
Treatment of Arnt-floxed/Mx1-Cre mice with pIpC resulted in
near-complete exon 6 deletion in liver and loss of ARNT mRNA
expression. The extent of recombination was also complete in the
thymus. Cre-mediated deletion in other tissues was incomplete and
ranged from 3070%. Other investigators also observed incomplete
recombination in extrahepatic tissues using two different transgenic
Mx1-Cre mice on different floxed gene backgrounds (11, 13). The reason
for this incomplete recombination using the Mx1-Cre transgene is not
clear. Interestingly, spontaneous recombination was found only in the
thymus and might be due to endogenous production of interferon. The
partial deletion product that resulted in removal of the PGK-neo was
also found in this tissue, but not in any other tissues of
Arnt-floxed/Mx1-Cre mice treated with pIpC. This result
would support the contention that recombination within a single cell is
complete and that the partial recombination detected in most tissues is
due to mixed populations of cells. Some cell types may not respond
because they lack the interferon receptor.
Complete disruption of ARNT expression results in loss of
AHR-stimulated gene activation in the liver by TCDD. These findings
definitively establish in an intact animal model that ARNT is required
for AHR function. In lung, Cre-mediated recombination resulted in loss
of more than 80% of ARNT mRNA expression, and yet full induction of
CYP1A1 mRNA by TCDD was found. These results suggest that ARNT levels
are in excess of AHR in this tissue and are in agreement with recent
studies in cell culture where it was found that ARNT was not limiting
(18). However, it should be noted that lung is a complex tissue;
certain cell types that have no AHR expression may have more complete
Arnt recombination than other AHR-expressing cells.
Alternatively, lung may contain a second heterodimerization partner for
AHR. Indeed, two other proteins, designated ARNT2 (19) and ARNT3 (20)
have been identified. However, ARNT2 is only expressed in adult
brain and kidneys, whereas ARNT3 is expressed in brain and skeletal
muscle. This is in contrast to ARNT that is ubiquitously expressed in
the adult mouse. All three proteins are expressed in embryos. In
contrast to the result in lung, in the heart, where ARNT expression is
also reduced by 80%, the extent of induction of CYP1A1 by TCDD is
reduced. Thus, ARNT levels may be limiting in heart tissue.
ARNT is also the putative dimerization partner for other bHLH-PAS
transcription superfamily members, such as the HIF-1
(3). Indeed, it
is thought that lack of ARNT in the developing embryo results in
disruption of HIF-1
-dependent angiogenesis (9, 10). To address
whether ARNT affects HIF-1
signal transduction in liver, mice were
administered CoCl2, an agent that is thought to
mimic hypoxia. Indeed, loss of ARNT expression selectively affected
HIF-1
-induced target genes. Heme oxygenase-1 mRNA was markedly
reduced, and glucose transporter-1 mRNA induction was abolished after
CoCl2 administration. However, induction of
aldolase A mRNA was not significantly abrogated by deletion of
Arnt. The reason for this variable response among putative
HIF-1
target genes is not known. One possible explanation is that
certain HIF-1
target genes may require only very small amounts of
the HIF-1
/ARNT heterodimer, which may be found if there is
incomplete deletion of Arnt in liver. In any case, these
data indicate that ARNT is also required for HIF-1
signal
transduction in the intact liver, thus confirming studies in cell
culture (21). It should be of interest to determine the role of ARNT in
relation to HIF-1
signal transduction in extrahepatic tissues such
as the central nervous system and blood vessels. Complete disruption of
Arnt in other tissues may reveal phenotypes that could lead
to the identification of new members of the basic helix-loop-helix/PAS
family that require ARNT for signal transduction. This will require
different Cre transgenes that can induce recombination and disruption
of ARNT expression in other tissues.
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MATERIALS AND METHODS
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Targeting Vector and Generation of ARNT Conditional-Null Mice
A mouse 129/Svj BAC genomic library (Genome Systems, St. Louis, MO) was screened with an oligonucleotide
probe corresponding to the mouse Arnt gene-coding sequence,
5'-cagaactgtcagacatggtacctacatgtagtg-3'. A 6.5-kb SacI
fragment containing the 5'-end of the bHLH exon of the Arnt
gene was isolated, subjected to restriction mapping, and used to make
the targeting construct (Fig. 1
). The targeting vector was made by
inserting the first loxP site into the EcoRI site of a
region 200 bp downstream of exon 6. This exon encodes the bHLH region
of ARNT (22). The second and the third loxP sites along with the
PGK-neo gene (loxP-neo-loxP cassette) were inserted, with the same
orientation as that of the first loxP site, in intron 5 using an
AvrII site located 1.5 kb upstream of the exon 6. The three
loxP sites flanked a 2-kb genomic region spanning the bHLH domain of
exon 6. The targeting vector contained 2.6 kb of homologous DNA
downstream (right arm) of the first loxP site and 2.2 kb of homologous
DNA upstream (left arm) of the loxP-neo-loxP cassette site. Gene
targeting was performed as previously described (23). All animal
studies were carried out under a protocol approved by the NIH animal
care and use committee. The linearized targeting vector was
electroporated into ES cells (RW4, Genome Systems) that
were maintained on subconfluent embryonic fibroblasts. G418-resistant
ES cell colonies were picked and expanded, and the ES cells were scored
for homologous recombination by Southern blotting using a 5'-probe
corresponding to the gene regions depicted in Fig. 1
. The targeted ES
cells were injected into C57BL/6 blastocysts, and the blastocysts were
transferred into pseudopregnant mothers. Highly chimeric mice were bred
with C57BL/6, and the F1 mice with germline
transmission of the loxP-targeted Arnt allele were bred with
homozygous Mx1-Cre mice (11). The F2 offspring
heterozygous for both the loxP-targeted Arnt
(Arnt-floxed) gene and the Cre transgene
(Cre+, Arnt-floxed/+) were mated with
heterozygous Arnt-floxed mice (+/+,
Arnt-floxed/+) to obtain F3 mice
carrying the Cre transgene as well as the homozygous
Arnt-floxed (Cre/+,
Arnt-floxed/Arnt-floxed), designated,
Arnt-floxed/Mx1-Cre mice.
PCR-Based Genotyping Assays
PCR primers used were ARNTYPE-F2 (5'-tgccaacatgtgccaccatgt-3'),
ARNTYPE-R2 (5'-gtgaggcagatttcttccatgctc-3'), Cre-F
(5'-aggtgtagagaaggcacttagc-3'), and Cre-R
(5'-ctaatcgccatcttccagcagg-3'). ARNTYPE-F2 and ARNTYPE-R2 flank the
3'-loxP site of the targeted allele and were used for routine
genotyping of Arnt. A 290-bp product was produced from the
wild-type allele, and a 340-bp product was generated from the targeted
allele due to the insertion of the loxP recognition site. Transmission
of the Mx1-Cre transgene was routinely monitored by PCR using primers
that produced a 478-bp product corresponding to nucleotides 1478 of
the bacteriophage P1 Cre gene. DNA was prepared from tail clips and
used to monitor transmission of the Arnt-floxed gene and the
Mx1-Cre gene. All amplifications were performed with 250 ng genomic DNA
using 1.25 U AmpliTaq DNA polymerase (Perkin-Elmer Corp.,
Norwalk, CT) in the presence of 2.5 mM
MgCl2, 200 µM of each
deoxy-NTP, and 1 µM of each primer in a final
volume of 25 µl. The following thermal-cycling profile was used for
all PCR reactions: 1 cycle of 5 min at 95 C; 30 cycles of 45 sec at 95
C, 45 sec at 60 C, and 45 sec at 72 C; and 1 cycle of 72 C for 6 min.
PCR products were separated on 2.5% agarose gels using 0.5 x TAE
(20 mM Tris-acetate and 2
mM
Na2EDTA·2H2O, pH 8.5) as
the running buffer and were visualized under UV light after staining
with ethidium bromide.
Analysis of Gene Expression
To examine the effect of disruption of the Arnt gene,
8-week-old male Arnt-floxed mice, with or without the
Mx1-Cre transgene, were administered 500-µg ip injections of pIpC
(Sigma, St. Louis, MO) dissolved in sterile saline every 3
days for a total of three injections. Control animals were injected
with saline alone. Five days after the final injection, the mice were
given a single 80 µg/kg ip injection of TCDD dissolved in
sterile-filtered corn oil. Control animals were given a single
injection of corn oil. For some experiments, mice were administered a
single 60 mg/kg sc injection of CoCl2
(Sigma) dissolved in sterile saline. Control animals were
injected with sterile saline alone. At 24 or 10 h after the TCDD
or CoCl2 injections, respectively, the mice were
killed by carbon dioxide asphyxiation. Tissues were harvested,
snap-frozen in liquid N2, and stored at -80 C
until use. Total RNA was prepared using TRIzol reagent (Life Technologies, Inc., Grand Island, NY) and analyzed by
electrophoresis on 1.1 M formaldehyde-containing
1% agarose gels. The separated RNA was transferred to GeneScreen Plus
membranes (NEN Life Science Products, Boston, MA) by
downward capillary transfer in the presence of 20 x SSC buffer (3
M NaCl and 0.3 M sodium
citrate, pH 7.0). After baking for 2 h at 80 C, the membranes were
immersed in Ultrahyb hybridization buffer (Ambion, Inc.,
Austin TX) and incubated for 2 h at 42 C. Full-length
complementary DNA (cDNA) probes for CYP1A1 (24), CYP1A2 (24), AHR (25),
and UGT1106 (26) were previously described. RT-PCR of mouse liver RNA
was used to produce a 2.4-kbp ARNT cDNA (forward,
5'-cgcggatccgatggcggcgactacagc-3'; reverse,
5'-ccggaattcctattcggaaaagggg-3') (27) and a 2.7-kbp HIF-1
cDNA
(forward, 5'-cgcggatccgcatggagggcgccggcgg-3'; reverse,
5'-ccggaattcctcagttaacttgatccaaagc-3') (28). To facilitate subcloning
of the PCR products, the primers contained BamHI and
EcoRI restriction sites at their 5'- and 3'-ends,
respectively. Total RNA was prepared from C57BL/6 mouse liver and was
used as a template for first strand cDNA synthesis using reverse
transcriptase. The PCR reaction was performed using Pfu DNA
polymerase under the following conditions: denature, 94 C, 30 sec;
primer anneal, 55 C, 1 min; primer extension, 72 C, 5 min. For
sequencing, the product of the second PCR was digested with
BamHI and EcoRI and subcloned into a pGEM-3Zf(+)
plasmid (Promega Corp., Madison, WI) to generate the
plasmid clones of each cDNA. RT-PCR of mouse liver RNA was used for
production of a 723-bp probe for the aldolase A mRNA (forward,
5'-aggaaccaatggcgagacaac-3'; reverse, 5'aagagagattcactggctgcgg-3')
(29), a 799-bp probe for glucose transporter-1 mRNA (forward,
5'-cgcttcctgctcatcaatcg-3'; reverse, 5'- tcttgtcactttggctggcac-3')
(30), and a 1059-bp probe for heme oxygenase-1 mRNA (forward,
5'-cctccagagtttccgcatacaac-3'; reverse, 5'ccaggcaagattctcccttacag-3')
(31) based upon the published cDNA sequences. Briefly, 1 µg total RNA
was reverse transcribed using Superscript II reverse transcriptase
(Life Technologies, Inc.) according to the manufacturers
instructions, using the indicated reverse primer. Twenty percent of the
cDNA synthesis reaction was used as a template for a PCR reaction
consisting of 2.5 U Vent DNA polymerase (New England Biolabs, Inc., Beverly, MA), 2.5 mM
MgCl2, 200 µM of each
deoxy-NTP, and 1 µM of each gene-specific
primer in a final volume of 50 µl. The following thermal-cycling
profile was used for all PCR reactions: 1 cycle of 5 min at 95 C; 30
cycles of 45 s at 95 C, 45 s at 60 C, and 45 s at 72 C;
and 1 cycle of 72 C for 6 min. PCR products were cloned into the
PCRII-Topo vector (Invitrogen, Carlsbad, CA), analyzed by
gel electrophoresis, and sequenced to confirm their identities. Probes
were 32P labeled by the random primer method
using Ready-to-Go DNA labeling beads (Amersham Pharmacia Biotech, Piscataway, NJ) and were added to the hybridization
buffer (1 x 106 cpm/ml), and incubation at
42 C was continued overnight. The blots were then washed once with
2 x SSC/1% SDS at 42 C for 15 min, twice with 0.1 x
SSC/1% SDS at 65 C, and once with 0.1 x SSC at room temperature.
After wrapping in plastic wrap, the blots were exposed to a
PhosphorImager screen cassette, and the signals were visualized using a
Storm 860 PhosphorImager system (Molecular Dynamics, Inc.,
Sunnyvale, CA).
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ACKNOWLEDGMENTS
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We thank Drs. R. Kuhn and K. Rajewski for providing the Mx1-Cre
mice, and B. Sauer for helpful suggestions during the course of these
studies.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Frank J. Gonzalez, Building 37, Room 3E-24, National Institutes of Health, Bethesda, Maryland 20892. E-mail: fjgonz{at}helix.nih.gov
C.J.S. was supported by a postdoctoral Fellowship from the Medical
Research Council of Canada.
1 S.T. and C.J.S. contributed equally to this work. 
Received for publication February 18, 2000.
Revision received June 19, 2000.
Accepted for publication June 28, 2000.
 |
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