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Murine DP genomic clones were isolated from a 129/Sv genomic DNA library (Stratagene) with the corresponding cDNA as a probe. The targeting vector was constructed by inserting the neomycin resistance gene (pMC1-neo, Stratagene) into a unique Nhe I site of the first coding exon; this insertion disrupts the DP gene in the sequence encoding the third transmembrane domain of the protein. The herpes simplex virus thymidine kinase gene was inserted upstream. The targeting vector was linearized and introduced into E14-1 embryonic stem cells from 129/Ola mice by electroporation. Clones resistant to both G418 and gancyclovir were isolated and screened for homologous recombination by PCR amplification. Recombination was then confirmed by Southern blot hybridization. Two lines of embryonic stem cells were injected into C57BL/6 blastocysts to generate chimeric male offspring, which were then mated with C57BL/6 females. Pups with an agouti coat were genotyped by Southern blot analysis for determination of germ line transmission. Polyadenylated RNA was prepared from various organs, which were rapidly removed and homogenized in 10 volumes of Trizol (Gibco-BRL) with a Polytron homogenizer. RT-PCR analysis was performed with 10 μg of polyadenylated RNA with the forward primer, 5′-TCGGTCTTTTATGTGCTCG-TG-3′, corresponding to amino acids 57 to 63 in the first coding exon, and the reverse primer, 5′-TCCACGTTACTTTGCTGGGAA-3′, corresponding to amino acids 346 to 353 in the second coding exon. Northern blot analysis was performed on 10 μg of polyadenylated RNA as described [H. Oida et al., FEBS Lett. 417, 53 (1997)].
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2 or BW245C was measured.
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0342735265
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note
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5 littermates.
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2+-free phosphate-buffered saline (PBS) containing 0.1% (w/v) bovine serum albumin and 0.05 mM EDTA. The BAL fluid obtained from each animal was pooled and centrifuged at 150g for 10 min at 4°C. Cell pellets were resuspended in the same solution, and the number of nucleated cells staining with Türk solution was counted. Smears of cell suspensions were prepared with the use of a Cytospin II cytocentrifuge, stained with May-Grüwald-Giemsa dye, and subjected to a differential count of at least 300 cells under a microscope.
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23
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Mice were anesthetized with sodium pentobarbital (80 mg/kg, ip), and the tail vein was cannulated. The animals were injected intravenously with pancuronium bromide (0.1 mg/kg) and ventilated with the aid of a rodent ventilator at a rate of 60 strokes per minute and a stroke volume of 0.6 ml of air supplemented with oxygen. Airway resistance was measured according to the overflow method described by H. Konzett and R. Rössler [Arch. Exp. Pathol. Pharmakol. 195, 71 (1940)] with the use of a Ugo Basil 7020 bronchospasm transducer connected to the tracheal cannula. Increasing doses of acetylcholine (31.25 to 2000 (μg/kg) were injected into the tail vein, and the changes in overflow volume were determined. The increase in respiratory overflow volume induced by acetylcholine was expressed as a percentage of the maximal overflow volume obtained by clamping the trachea! cannula.
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0342300215
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note
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-1- mice 8 hours after the last inhalation of OVA, and the concentrations of IL-4, IL-5, IL-13, and IFN-γ in the fluid were determined by enzyme-linked immunosorbent assay.
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25
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0342300214
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note
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Twenty-four hours after the last OVA inhalation, mice were anesthetized with sodium pentobarbital, and 10% (v/v) formalin in PBS was infused into the lung for fixation. The lung was then sectioned and stained either with hematoxylin and eosin or with periodic acid-Schiff reagent.
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-1- mice. However, for excessive OVA challenges that induced infiltration of ∼70 × 10s cells in wild-type mice, a similar number of cells was detected in the BAL fluid of DP-1- mice.
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Statistical significance was evaluated by one-way analysis of variance followed by Student's t test for unpaired values.
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39
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0343567079
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note
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We thank K. Ishikawa, Y. Kataoka, K. Deguchi, and T. Obata for technical assistance; T. Arai and H. Nose for secretarial assistance; T. Tanaka and M. Kitaichi for discussions; and O. Hayaishi for encouragement. Supported in part by grants from the Uehara Memori, Foundation and the Smoking Research Foundation.
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