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{"project":"2_test","denotations":[{"id":"16221973-2440339-77403413","span":{"begin":2582,"end":2584},"obj":"2440339"},{"id":"16221973-12519945-77403414","span":{"begin":4276,"end":4278},"obj":"12519945"},{"id":"16221973-390497-77403415","span":{"begin":5093,"end":5095},"obj":"390497"},{"id":"16221973-8467794-77403416","span":{"begin":5818,"end":5820},"obj":"8467794"},{"id":"16221973-8467794-77403417","span":{"begin":6699,"end":6701},"obj":"8467794"},{"id":"16221973-8467794-77403418","span":{"begin":6832,"end":6834},"obj":"8467794"},{"id":"16221973-8467794-77403419","span":{"begin":7593,"end":7595},"obj":"8467794"},{"id":"16221973-4705382-77403420","span":{"begin":8483,"end":8485},"obj":"4705382"},{"id":"16221973-38242-77403421","span":{"begin":9347,"end":9349},"obj":"38242"}],"text":"MATERIALS AND METHODS\n\nGeneration of Mtf1 conditional knockout mice and liver-specific deletion\nMtf1 conditional knockout mice were generated in collaboration with Dr Michael Leviten (San Carlos, CA). Two genomic clones containing exons 3 to 6 of Mtf1 were used to construct a gene targeting vector for homologous recombination (Supplementary Data). A neomycin resistance cassette (PGK-neo) flanked by two loxP sites was cloned into the SacI site 5′ of exon 3 of Mtf1, the third loxP site was cloned into the ScaI site 3′ of exon 4. A thymidine kinase (TK) cassette was inserted in the HpaI site 3′ of exon 6. 129 ES cells were electroporated with the linearized targeting vector, selected in the presence of G418 and FIAU, and screened for correct integration events by PCR and Southern blot analysis. Transient expression of Cre recombinase led to removal of the PGK-neo cassette, and mice carrying the modified Mtf1loxP allele were generated by injection of positive clones into C57Bl/6 blastocysts and subsequent crosses. Homozygous conditional knockout animals (Mtf1loxP/loxP) were crossed with the Cre recombinase transgenic line Mx-cre (a gift from Prof. Michel Aguet) to obtain an inducible, liver-specific Mtf1 knockout line. The mice were genotyped by PCR using the following primers (Microsynth): Cre recombinase: 5′-CTATCCAGCAACATTTGGGCCAGC-3′; 5′-CCAGGTTACGGATATAGTTCATGAC-3′, Mtf1loxP or wild-type allele: 5′-CACACCCAGTTTGTGTATGTCTTC-3′; 5′-CAGTCACAAGCAAATTACCAAACACTGCC-3′.\n\nAnimal treatment\nAt 8 weeks of age, male Mtf1loxP/loxP mice harboring the Mx-cre transgene (Mtf1loxP/loxP Mx-cre, abbr.: Mtf1Mx-cre) and control littermates without transgene (Mtf1loxP/loxP, abbr.: Mtf1loxP) received four intraperitoneal injections each of 300 µg synthetic double-stranded RNA polyinosinic–polycytidylic acid [pI–pC; Sigma; in a volume of 60 µl phosphate-buffered saline (PBS)] at 3 day intervals. Only in the case of DNA-binding studies with MRE sequences from MTF-1 target gene candidates, the control mice received no pI–pC injections. For experiments with metal treatment, mice received 2 days after the last pI–pC treatment a subcutaneous (s.c.) injection of either 20 µmol/kg body weight CdSO4 (2 mM CdSO4 in H2O; cadmium treatment) or 10 ml/kg body weight H2O (mock treatment) 6 h before sacrificing them.\n\nMicroarray analysis and data processing\nTotal RNA was isolated from liver tissue of pI–pC-induced, mock- or cadmium-treated Mtf1Mx-cre and Mtf1loxP mice (n = 3 per genotype and respective treatment; all male) essentially as described by Chomczynski and Sacchi (27).\nGene expression analysis was performed in the Functional Genomics Center Zurich using GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix) according to the manufacturer's instructions and the following reagents and conditions. cDNA was synthesized with SuperScript™ Double-Stranded cDNA Synthesis Kit (Invitrogen), using 15 µg total RNA. In vitro transcription was performed with BioArray™ High Yield™ RNA Transcript Labeling Kit (Enzo) and 3.5 to 6 µg of each cDNA. Clean-up of both cDNA and cRNA samples was done using GeneChip® Sample Clean-up Module (Affymetrix). For the automated washing and staining in the Affymetrix fluidics station 450, the protocol EukGE-Ws2v4_450 was used. The probe arrays were scanned with the Affymetrix GS 3000 scanner. Raw data are available at ArrayExpress (, accession number E-MEXP-438).\nData analysis was performed with GeneSpring 6.1 software (Silicon Genetics), applying a significance level P ≤ 0.05. Furthermore, multiple testing correction was used in addition to obtain cadmium-responsive, MTF-1-independent genes. Genes were considered to be differentially expressed if there was at least a 2-fold difference in expression levels of the compared experimental groups (genotype and respective treatment). The result was considered reliable if signal values for the respective gene were scored ‘present’ at least for all mice in one experimental group or for two mice in each of two groups.\nTo screen for the presence of the MRE core consensus sequence TGCRCNC in the promoter region, the upstream sequences of the respective genes were obtained from the University of California Santa Cruz (UCSC) Genome Browser Database (; October/November 2004) (28).\n\nRT–PCR\nAll RT–PCRs were performed with QIAGEN® OneStep RT–PCR Kit (QIAGEN) according to the manufacturer's instructions, using 150–200 ng DNase I-digested total RNA (RNA isolation see microarray analysis). The reactions were carried out using the following primers: Csrp1: 5′-TTCCGATGTGCCAAGTGTGGC-3′; 5′-AGTAGAGAGTGGACATTCAGC-3′, hypoxanthin-guanin-phosphoribosyltransferase (Hprt): 5′-GCTGGTGAAAAGGACCTCTCG-3′; 5′-CCACAGGACTAGGACACCTGC-3′, Mtf1: 5′-GTGACTTTTGAGACTGTACTGAGTG-3′; 5′-CATGCCAAGAAACATTGAAGGTG-3′, Ndrg1: 5′-AGATACACAACAACGTGGAGG-3′; 5′-TGTGCGAGCGGCTTCGGGGGC-3′, Sepw1: 5′-TAGAGGCAGGGTCCTGAAAGC-3′; 5′-ACACCTGGAAACATGGCTGCC-3′, Slc39a10: 5′-GCTGTGGCTGGTAGTAAAAGC-3′; 5′-GTGGCATGGGATGTAAACAGC-3′.\n\nS1 nuclease mapping of transcripts (S1 analysis)\nS1 analysis was performed as previously described (29), using 100 µg DNase I-digested total RNA (RNA isolation see microarray analysis). The gels were developed using PhosphorImager (Molecular Dynamics). S1 analysis was done with the following 32P-labeled oligonucleotides: Hprt S1: 5′-TCTTCAGTCTGATAAAATCTACAGTCATAGGAATGGATCTATCACTATTTCTATTCAGTGATTACATTAAAG-3′, Sepw1 S1: 5′-TTCAACCGGGAACACCTGGAAACATGGCTGCCTGTCTTCTTGAAGTCTTGAGGTGGAAAGGGAAAGCAAAGCAGGAGGGTTTTCCCACCC-3′.\n\nElectrophoretic mobility shift assay (EMSA)\nProtein was extracted from liver tissue with T-PER™ Tissue Protein Extraction Reagent (Pierce) according to the manufacturer's instructions, using a 1:10 ratio of mouse tissue (mg) to T-PER reagent (µl).\nEMSA was essentially performed as previously described (10). All binding reactions were carried out by incubating 2–5 fmol 32P-end-labeled double-stranded oligonucleotides with 100 to 130 µg liver protein extract. For competition experiments, 5 pmol of unlabeled competitor oligonucleotide was added to the binding reaction before addition of the extract. All EMSA gels were developed using PhosphorImager (Molecular Dynamics). The following oligonucleotides were annealed and used for the reactions: Csrp1 MRE1: 5′-GGAAACAAAACGGCGCGCACTCCGGCGC-3′; 5′-GGCTGCGC CGGAGTGCGCGCCGTTTTGT-3′, Csrp1 MRE2: 5′-TGTTGTGGTGCAGTGTGCAAAGCCTAC-3′; 5′-ACCAGTAGGCTTTGCACACTGCACCAC-3′, Csrp1 MRE3: 5′-GAGATCGCCATAGGGTGCAAAGAGAAG-3′; 5′-GTGACTTCTCTTTGCACCCTATGGCGA-3′, Csrp1 MRE4: 5′-TGTCTTATTCTGGAGTGCAAGTTAGTC-3′; 5′-AGGGGACTAACTTGCACTCCAGAATAA-3′, Gal4: 5′-TCCGGAGGACTGTCCTCCGG-3′; 5′-GCCGGAGGACAGTCCTCCGG-3′, MREd [MRE derived from mouse Mt1 promoter (10)]: 5′-CGAGGGAGCTCTGCACTCCGCCCGAAAAGTG-3′; 5′-TCGACACTTTTCGGGCGGAGTGCAGAGCTCCCTCGAGCT-3′, MRE-s [synthetic MRE consensus sequence (10)]: 5′-CGAGGGAGCTCTGCACACGGCCCGAAAAGTG-3′; 5′-TCGACACTTTTCGGGCCGTGTGCAGAGCTCCCTCGAGCT-3′, Ndrg1 MRE1: 5′-CAGCCCAGGCAGGGTGCAGCACGAG-3′; 5′-CCGCCTCGTGCTGCACCCTGCCTGG-3′, Ndrg1 MRE2: 5′-CACACGTTCGCTGCACACGCCGCGG-3′; 5′-GGGACCGCGGCGTGTGCAGCGAACG-3′, Ndrg1 MRE3,4: 5′-GGAGTCCTTATGCACACGCGCACGAGCGCGCACGGGCAC-3′; 5′-TGGTGTGCCCGTGCGCGCTCGTGCGCGTGTGCATAAGGAC-3′, Sepw1 MRE1: 5′-GAGGCAGTCGGCTGTGCGCACGGCCCCACGCTC-3′; 5′-CTCTGAGCGTGGGGCCGTGCGCACAGCCGACTGC-3′, Sepw1 MRE2: 5′-ATGGTTTTGGGGGTGCGCAGGGGGTCTG-3′; 5′-CGACAGACCCCCTGCGCACCCCCAAAAC-3′, Slc39a10 MRE1: 5′-GAATACACGACTGGGTGCAGCCGGGGTTTGG-3′; 5′-GGTACCAAACCCCGGCTGCACCCAGTCGTGTA-3′, Slc39a10 MRE2: 5′-GCGGAGAGGAGATGCACACGGCACTCG-3′; 5′-CACTCGAGTGCCGTGTGCATCTCCTCT-3′, Specificity protein 1 (Sp1) binding sequence (10): 5′-CGAGGCCCCGCCCAG-3′; 5′-TCGACTGGGCGGGGCCTCGAGct-3′.\n\nCell culture\nPrimary embryonic fibroblasts were isolated from a 12.5 day old Mtf1loxP mouse embryo and grown in DMEM supplemented with 10% fetal bovine serum (ICN), 100 U/ml penicillin–streptomycin (Gibco BRL) and 2 mM l-glutamine (Gibco BRL). 100 mm plates with primary cells were transfected with 10 µg of an expression plasmid coding for simian virus 40 (SV40) large T antigen driven by the cytomegalovirus (CMV) promoter, using lipofectamine™ reagent (Invitrogen) according to the manufacturer's instructions. Cell foci were isolated and the immortalized mouse embryonic fibroblast cell line ckoC was derived from one of them. The Mtf1loxP genotype of this line as well as the genomic integration of the T antigen were confirmed by PCR. 100 mm plates with these cells were further transfected by the calcium phosphate method (30) with 19.6 µg of an expression plasmid for Cre recombinase driven by the CMV promoter and 0.4 µg of an expression plasmid for the neomycin resistance gene under the control of the TK promoter. Stably transfected cells were selected in the presence of 0.4 µg/µl G418 (Calbiochem), isolated clones of resistant cells were harvested and grown independently, and the expression of Cre recombinase and excision of exons 3 and 4 of Mtf1 were analyzed by RT–PCR. The cell lines delC19, delC21 and delC23 with a deletion of Mtf1 were chosen for further experiments.\n\nCytotoxicity assay\nSamples of 1 × 104 cells/well were plated in 96-well tissue culture plates and allowed to adhere for 24 h. The cells were then pre-incubated for 24 h in medium containing 0, 5, 10, 25 or 50 µM l-buthionine-[S,R]-sulfoximine (BSO) (Sigma), a drug that inhibits glutathione synthesis (31). Later, cells were exposed to 0, 5, 10 or 20 µM CdCl2 in the specified pre-incubation medium for an additional 24 h. Cytotoxicity was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid)-based Cell Proliferation Kit I (Roche) according to the manufacturer's instructions."}

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