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{alpha}

Shuhei Tomita1, Christopher J. Sinal1, Sun Hee Yim and Frank J. Gonzalez

Laboratory of Metabolism National Cancer Institute Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{gamma} 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{alpha} activity. These studies establish a critical role for ARNT in AHR and HIF-1{alpha} signal transduction in the intact mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}) (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{alpha} signal transduction in the intact animal.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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.



View larger version (19K):
[in this window]
[in a new window]
 
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.

 
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. 2Go). 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-{gamma} 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).



View larger version (24K):
[in this window]
[in a new window]
 
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.

 
In Arnt-floxed mice treated with pIpC, the Arnt exon 6 was deleted more than 95% in liver and thymus (Fig. 3Go). 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.



View larger version (41K):
[in this window]
[in a new window]
 
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. 1Go. 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.

 
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. 4Go). 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-{gamma} 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 30–50% of ARNT mRNA remained after pIpC treatment. Expression of ARNT mRNA in intestine was not significantly lower despite about 30% deletion in this tissue.



View larger version (57K):
[in this window]
[in a new window]
 
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.

 
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. 5Go). 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. 6Go). 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.



View larger version (53K):
[in this window]
[in a new window]
 
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.

 


View larger version (32K):
[in this window]
[in a new window]
 
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.

 
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. 7Go). The extent of induction by TCDD was not changed in kidney, intestine, or lung.



View larger version (37K):
[in this window]
[in a new window]
 
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.

 
Loss of HIF-1{alpha} Function in ARNT Conditional- Null Mice
To further examine the functional consequences resulting from loss of ARNT expression, target genes for HIF-1{alpha} 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. 8Go). 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{alpha}-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. 8Go). In contrast, only a modest loss of aldolase A mRNA induction occurred as a result of Arnt deletion. Analysis of HIF-1{alpha} 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{alpha}. 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.



View larger version (56K):
[in this window]
[in a new window]
 
Figure 8. Expression of HIF-1{alpha} mRNA and mRNA Derived from Putative HIF-1{alpha} 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Standard gene targeting of Arnt in ES cells results in embryonic lethality that is thought to be due to disruption of the HIF-1{alpha}-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-{gamma} 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 30–70%. 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{alpha} (3). Indeed, it is thought that lack of ARNT in the developing embryo results in disruption of HIF-1{alpha}-dependent angiogenesis (9, 10). To address whether ARNT affects HIF-1{alpha} 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{alpha}-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{alpha} target genes is not known. One possible explanation is that certain HIF-1{alpha} target genes may require only very small amounts of the HIF-1{alpha}/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{alpha} 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{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go. 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 1–478 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{alpha} 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 manufacturer’s 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).


    ACKNOWLEDGMENTS
 
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
 
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. Back

Received for publication February 18, 2000. Revision received June 19, 2000. Accepted for publication June 28, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Rowlands JC, Gustafsson JA 1997 Aryl hydrocarbon receptor-mediated signal transduction. Crit Rev Toxicol 27:109–134[Medline]
  2. Sogawa K, Fujii-Kuriyama Y 1997 Ah receptor, a novel ligand-activated transcription factor. J Biochem 122:1075–1079[Abstract]
  3. Semenza GL 2000 HIF-1: mediator of physiological, pathophysiological responses to hypoxia. J Appl Physiol 88:1474–1480[Abstract/Free Full Text]
  4. Ema M, Morita M, Ikawa S, Tanaka M, Matsuda Y, Gotoh O, Saijoh Y, Fujii H, Hamada H, Kikuchi Y, Fujii-Kuriyama Y 1996 Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis. Mol Cell Biol 16:5865–5875[Abstract]
  5. Whitmore D, Sassone-Corsi P, Foulkes NS 1998 PASting together the mammalian clock. Curr Opin Neurobiol 8:635–641[CrossRef][Medline]
  6. Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, Eichele G, Lee CC, Bradley A 1999 The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169–173[CrossRef][Medline]
  7. Rowe A 1997 Retinoid X receptors. Int J Biochem Cell Biol 29:275–278[CrossRef][Medline]
  8. Sogawa K, Nakano R, Kobayashi A, Kikuchi Y, Ohe N, Matsushita N, Fujii-Kuriyama Y 1995 Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation. Proc Natl Acad Sci USA 92:1936–1940[Abstract]
  9. Maltepe E, Schmidt JV, Baunoch D, Bradfield CA, Simon MC 1997 Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 386:403–407[CrossRef][Medline]
  10. Kozak KR, Abbott B, Hankinson O 1997 ARNT-deficient mice and placental differentiation. Dev Biol 191:297–305[CrossRef][Medline]
  11. Kuhn R, Schwenk F, Aguet M, Rajewsky K 1995 Inducible gene targeting in mice. Science 269:1427–1429[Medline]
  12. Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J, Schwenk F 1996 Conditional gene targeting. J Clin Invest 98:600–603[Free Full Text]
  13. Raabe M, Veniant MM, Sullivan MA, Zlot CH, Bjorkegren J, Nielsen LB, Wong JS, Hamilton RL, Young SG 1999 Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J Clin Invest 103:1287–1298[Abstract/Free Full Text]
  14. Rohlmann A, Gotthardt M, Hammer RE, Herz J 1998 Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J Clin Invest 101:689–695[Abstract/Free Full Text]
  15. Kimura S, Gonzalez FJ, Nebert DW 1986 Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues. Mol Cell Biol 6:1471–1477[Medline]
  16. Goldberg MA, Glass GA, Cunningham JM, Bunn HF 1987 The regulated expression of erythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci USA 84:7972–7976[Abstract]
  17. Beru N, McDonald J, Lacombe C, Goldwasser E 1986 Expression of the erythropoietin gene. Mol Cell Biol 6:2571–2575[Medline]
  18. Pollenz RS, Davarinos NA, Shearer TP 1999 Analysis of aryl hydrocarbon receptor-mediated signaling during physiological hypoxia reveals lack of competition for the aryl hydrocarbon nuclear translocator transcription factor. Mol Pharmacol 56:1127–1137[Abstract/Free Full Text]
  19. Hirose K, Morita M, Ema M, Mimura J, Hamada H, Fujii H, Saijo Y, Gotoh O, Sogawa K, Fujii-Kuriyama Y 1996 cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt). Mol Cell Biol 16:1706–1713[Abstract]
  20. Takahata S, Sogawa K, Kobayashi A, Ema M, Mimura J, Ozaki N, Fujii-Kuriyama Y 1998 Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock. Biochem Biophys Res Commun 248:789–794[CrossRef][Medline]
  21. Semenza GL 2000 Expression of hypoxia-inducible factor 1: mechanisms, consequences. Biochem Pharmacol 59:47–53[CrossRef][Medline]
  22. Wang F, Gao JX, Mimura J, Kobayashi A, Sogawa K, Fujii-Kuriyama Y 1998 Structure and expression of the mouse AhR nuclear translocator (mArnt) gene. J Biol Chem 273:24867–24873[Abstract/Free Full Text]
  23. Hogan B, Lyons K 1988 Gene targeting. Getting nearer the mark. Nature 336:304–305[CrossRef][Medline]
  24. Gonzalez FJ, Mackenzie PI, Kimura S, Nebert DW 1984 Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1–450 and P3–450. Gene 29:281–292[CrossRef][Medline]
  25. Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS, Kimura S, Nebert DW, Rudikoff S, Ward JM, Gonzalez FJ 1995 Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268:722–726[Medline]
  26. Kong AN, Ma M, Tao D, Yang L 1993 Molecular cloning of two cDNAs encoding the mouse bilirubin/phenol family of UDP-glucuronosyltransferases (mUGTBr/p). Pharm Res 10:461–465[CrossRef][Medline]
  27. Hoffman EC, Reyes H, Chu FF, Sander F, Conley LH, Brooks BA, Hankinson O 1991 Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252:954–958[Medline]
  28. Wang GL, Jiang BH, Rue EA, Semenza GL 1995 Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 92:5510–5514[Abstract]
  29. Mestek A, Stauffer J, Tolan DR, Ciejek-Baez E 1987 Sequence of a mouse brain aldolase A cDNA. Nucleic Acids Res 15:10595[Medline]
  30. Kaestner KH, Christy RJ, McLenithan JC, Braiterman LT, Cornelius P, Pekala PH, Lane MD 1989 Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3–L1 adipocytes. Proc Natl Acad Sci USA 86:3150–3154[Abstract]
  31. Alam J, Cai J, Smith A 1994 Isolation and characterization of the mouse heme oxygenase-1 gene. Distal 5' sequences are required for induction by heme or heavy metals. J Biol Chem 269:1001–1009[Abstract/Free Full Text]