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An ∼ 12-kb Bam HI-Xho I fragment of the NK-1R gene, including exon 1 and ∼7 kb of the 5′ sequence, was cloned from a mouse Sv 129 genomic library. Through sequential cloning steps, most of exon 1 was deleted, including the sequence from the Sac I site ∼80 nucleotides downstream from the TATAA sequence to the Stu I site just before the end of the exon 1 coding sequence, and replaced with the genes for β-galactosidase and neomycin resistance. The construct was transfected into J1 embryonic stem cells and doubly selected for integration with G418 and gancydovir. Homologous recombination was determined by restriction analyses of independent clones, with both flanking and internal probes (probes A and B, respectively). Three clones among ∼100 screened were positive for homologous recombination into the NK-1R locus. They were injected into mouse blastocysts and resulted in eight chimeric offspring, which were bred with C57BL/6 mice for germline transmission.
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Littermate mice of ∼30 g were used. Capsaicin was dissolved in acetone (10 mg/ml) and 25 μlwas applied topically to both surfaces of one ear. Acetone alone was applied to the contralateral ear. After 30 min, ear thickness was measured with dial calipers, and 6-mm punch biopsies were weighed [H. Inoue, N. Nagata, Y. Koshihara, Br. J. Pharmacol. 110, 1614 (1993); C. R. Mantione and R. Rodriguez, ibid. 99, 516 (1990)].
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0025100773
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Littermate mice of ∼30 g were used. Capsaicin was dissolved in acetone (10 mg/ml) and 25 μlwas applied topically to both surfaces of one ear. Acetone alone was applied to the contralateral ear. After 30 min, ear thickness was measured with dial calipers, and 6-mm punch biopsies were weighed [H. Inoue, N. Nagata, Y. Koshihara, Br. J. Pharmacol. 110, 1614 (1993); C. R. Mantione and R. Rodriguez, ibid. 99, 516 (1990)].
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Mice matched for sex and age (12 to 14 weeks) were used for studies of immune complex-induced lung injury [J. S. Warren et al., J. Clin. Invest. 84, 1873 (1989)]. Rabbit antibody to chicken egg albumin (300 μg) was injected intratracheally, followed by injection of chicken egg albumin (20 mg/kg) into a tail vein. Some animals were tracheostomized after they were killed, and BAL was performed with saline. The recovered BAL fluid was assessed for differential cell counts and cytokine measurements. Permeability indexes were determined by comparing the amount of Evans blue in BAL fluid with the amount present in 1 ml of plasma. No statistical differences were observed between wild-type animals and heterozygotes in this assay. Paraffin sections were prepared for histologic analyses after inflation of the lung with 10% neutral buffered formalin. All animal studies were performed according to institutional NIH guidelines for animal use and care.
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We thank B. Koller for support, advice, encouragement, and blastocyst injections; L. Kobzik for blinded histological analyses; C. De Dios for technical assistance; and M. Soares for help with enzyme-linked immunosorbent assay measurements. Supported by NIH grants HL36162 and HL51366, the Rubenstein-Cable Cystic Fibrosis Research Fund at the Perlmutter Laboratory, the Medical Research Council of Canada (C.R.B.), and Deutsche Forschungsgemeinschaft (U.E.H.).
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