Animals. 1 hybrid (n = 40), (A/J ? C3H/HeJ) F1 hybrid (n = 40), (A/J ? C3H/HeJ) Fdos intercross (n = 104), and [(A/J ? C3H/HeJ) ? C3H/HeJ] backcross (n = 369) mice 5–6 wk of age were purchased from Jackson Laboratory (Bar Harbor, ME). To minimize environmental effects, the mice were housed in isolation cages under VAF conditions. The mice were acclimatized for 10–14 days after transport and allowed free access to commercial pelleted mouse food and water, which were autoclaved to ensure sterility. VAF status was routinely confirmed by testing blood samples taken from sentinel animals. Because age (6, 21), gender (11), and airway infections (18) can alter airway responsiveness, only male, 8-wk-old mice housed in a barrier facility were studied.
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Phenotype analysis. The methodology for measuring airway responsiveness was as originally described by Martin et al. (14) and modified by De Sanctis and co-workers (3, 4). Dose-response curves to methacholine were obtained by administering sequentially increasing doses of methacholine (acetyl-?-methylcholine chloride; 33–3,300 ?g/kg iv; Sigma, St. Louis, MO) with a 0.1-ml Hamilton (Reno, NV) microsyringe. The volume of fluid injected with each dose produced no measurable physiological effects.RL was determined with the use of signals derived from transpulmonary pressure and lung volume (14). Each animal’s dose-response curve was log transformed and then subjected to regression analysis to calculate the dose required for a 1.50-fold increase inR150RL). Because the doses of methacholine are given in geometrically increasing amounts, it is common to log transform this index. QTL analysis was also carried out with the individualRL values (as a percentage of baseline) for the 330 and 1,000 ?g/kg doses from 369 backcross progeny.
DNA preparing. Each other kidneys had been very carefully got rid of, snap-suspended in the liquids nitrogen, and you will after that stored in a ?80°F freezer. Purified genomic DNA was extracted from one of the kidneys that have a DNA extraction equipment (Stratagene, Los angeles Jolla, CA). Spectrophotometric readings of DNA trials was basically delivered to be certain that their purity (ratio of absorbance in the 260 nm to this within 280 nm) and also to quantitate (absorbance from the 260 nm) the fresh new DNA attention.
Genotype analysis. Genotype analysis was performed with mouse simple sequence length polymorphic (SSLP) markers as previously described (5). Initially, a panel of between 81 and 97 SSLP markers, distributed at ?15-centimorgan (cM) intervals throughout the genome, was used to genotype 232 of the phenotypically most extreme backcross progeny. Because we identified a linkage on chromosome 6 in an area previously reported by others (8), four markers around the peak of the QTL on chromosome 6 were used to type all 369 backcross progeny. Primers were purchased from Research Genetics. PCR amplification was performed in 10 mM Tris, pH 8.3, 45 mM KCl, 1.5 mM MgCl2, and 0.2 mM each dATP, dCTP, dTTP, and dGTP. PCR was performed for 35 cycles at 94°C for 15 s, 58°C for 45 s, and 72°C for 45 s. Product size was analyzed by electrophoresis with 3.5% MetaPhor agarose and ethidium bromide staining.
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Statistical and linkage analysisputations were performed with the JMP 3.1.5 (SAS Institute, Cary, NC) statistical package. A Tukey-Kramer honestly significant difference test was used to assess differences between the parental strains and individual crosses. For nonparametric data, differences between groups were analyzed by the Wilcoxon rank sum test. Where appropriate, a Shapiro-Wilk W-test was used to assess normality. Standard ANOVA, including cross-terms for two-way Sacramento local hookup free interactions, was used to evaluate possible interactions between candidate QTLs (chromosomes 6 and 7) and the mouse airway responsiveness phenotypes (330 and 1,000 ?g/kg and log ED150RL). QTL analysis was carried out with the Map Manager QT computer package from the genotype data and the following airway responsiveness phenotypes: 1) the response ofRL to individual methacholine doses of 100, 330, and 1,000 ?g/kg and2) the log ED150RLvalues computed from the dose-response curves. The likelihood ratio statistic that Map Manager QT calculates for QTL mapping is a LOD score based on a natural logarithm. Conventional base 10 LOD scores were calculated from the likelihood ratio statistic values by multiplication with a constant (0.217). Results, expressed as means ± SD unless otherwise stated, were considered significant atP < 0.05.