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type and Cftr null mice on two different genetic backgrounds were used in this study. One group was congenic on the C57Bl/6 (B6) background, as previously described [21]. The other group was on a mixed background of about 95% B6 and 5% 129/Sv (129). The mice on the mixed background originated as part of a recently published study [20] as follows. Mice carrying a targeted mutation of the gastrin gene on a mixed B6/129 background [51] were bred for eight generations onto the B6 background. The gastrin(+/-) mice were then crossed for six generations with Cftr(+/-) mice congenic on the B6 background. A genome scan at this point showed that the mice were about 95% B6 and 5% 129 (see below). These mice were bred to obtain mice wild type for both gastrin alleles and either Cftr homozygous wild type [Cftr(+/+)] or Cftr homozygous null [Cftr(-/-)].\nMice were genotyped at 2 weeks of age by PCR as previously described [21]. Unless otherwise stated, mice were maintained on a complete elemental liquid diet (Peptamen; Nestle Deerfield, IL) to prevent intestinal obstruction that occurs in CF mice [52]. Wild type littermates were maintained on the same diet. In some experiments, 8 week old mice were transferred onto solid mouse chow instead of Peptamen, and survival was recorded. Mice with apparent distress were sacrificed and survival on chow was recorded as the following day. Male and female mice were used at 6–16 weeks of age. Mice were kept in a specific pathogen-free facility in barrier-top cages. All procedures were approved by the University of Kansas Medical Center IACUC.\n\nGenetic background determination\nThe Genome Scanning Service of The Jackson Laboratory (Bar Harbor, ME) was used to determine the contributions of C57Bl/6 and 129/Sv strains in the interbred mice. Pieces of mouse tail were sent to The Jackson Laboratory for simple sequence length polymorphism (SSLP) PCR analysis with the DMit primers specific for B6 and 129 strain alleles . The SSLP panel consists of 108 mapped markers designed to distinguish between B6 and 129 strains. The markers are spaced 12–13 cM apart and span the nineteen autosomes.\n\nMeasurement of gene expression\nTotal RNA was extracted from the entire small intestine as previously described [21]. Quantitative, real-time RT-PCR was used to measure expression of specific genes using the previously described primers [21]. Values were normalized to GAPDH mRNA and expression of this housekeeping gene is not altered in the CF mouse small intestine [21]. Expression of the major intestinal mucin, Muc2, was also measured using the forward (5'-GAC TTC GAT GGA CAC TGC TC-3') and reverse (5'-CAC GGT GTT TAT CTA CCA AC-3') primers.\n\nHistology\nThe small intestine was flushed with phosphate buffered saline and immersion fixed overnight in 4% paraformaldehyde. The tissues were then prepared for paraffin embedding and sectioning by a commercial service (HSRL, Woodstock, VA). Sections (5 μm) were stained with periodic acid Schiff's (PAS) for neutral mucins.\n\nStatistics\nGene expression and body weight data were compared by ANOVA with a post-hoc Tukey's analysis (Systat software, Chicago, IL). Survival data were analyzed by a log-rank test for P values (PEPI software, ). The distributions of genotypes of pups surviving to weaning from breeding Cftr(+/-) mice were compared to that expected by Mendelian genetics using Chi-square analysis. For all statistical tests, P \u003c 0.05 was considered significant."}

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One group was congenic on the C57Bl/6 (B6) background, as previously described [21]. The other group was on a mixed background of about 95% B6 and 5% 129/Sv (129). The mice on the mixed background originated as part of a recently published study [20] as follows. Mice carrying a targeted mutation of the gastrin gene on a mixed B6/129 background [51] were bred for eight generations onto the B6 background. The gastrin(+/-) mice were then crossed for six generations with Cftr(+/-) mice congenic on the B6 background. A genome scan at this point showed that the mice were about 95% B6 and 5% 129 (see below). These mice were bred to obtain mice wild type for both gastrin alleles and either Cftr homozygous wild type [Cftr(+/+)] or Cftr homozygous null [Cftr(-/-)].\nMice were genotyped at 2 weeks of age by PCR as previously described [21]. Unless otherwise stated, mice were maintained on a complete elemental liquid diet (Peptamen; Nestle Deerfield, IL) to prevent intestinal obstruction that occurs in CF mice [52]. Wild type littermates were maintained on the same diet. In some experiments, 8 week old mice were transferred onto solid mouse chow instead of Peptamen, and survival was recorded. Mice with apparent distress were sacrificed and survival on chow was recorded as the following day. Male and female mice were used at 6–16 weeks of age. Mice were kept in a specific pathogen-free facility in barrier-top cages. All procedures were approved by the University of Kansas Medical Center IACUC.\n\nGenetic background determination\nThe Genome Scanning Service of The Jackson Laboratory (Bar Harbor, ME) was used to determine the contributions of C57Bl/6 and 129/Sv strains in the interbred mice. Pieces of mouse tail were sent to The Jackson Laboratory for simple sequence length polymorphism (SSLP) PCR analysis with the DMit primers specific for B6 and 129 strain alleles . The SSLP panel consists of 108 mapped markers designed to distinguish between B6 and 129 strains. The markers are spaced 12–13 cM apart and span the nineteen autosomes.\n\nMeasurement of gene expression\nTotal RNA was extracted from the entire small intestine as previously described [21]. Quantitative, real-time RT-PCR was used to measure expression of specific genes using the previously described primers [21]. Values were normalized to GAPDH mRNA and expression of this housekeeping gene is not altered in the CF mouse small intestine [21]. Expression of the major intestinal mucin, Muc2, was also measured using the forward (5'-GAC TTC GAT GGA CAC TGC TC-3') and reverse (5'-CAC GGT GTT TAT CTA CCA AC-3') primers.\n\nHistology\nThe small intestine was flushed with phosphate buffered saline and immersion fixed overnight in 4% paraformaldehyde. The tissues were then prepared for paraffin embedding and sectioning by a commercial service (HSRL, Woodstock, VA). Sections (5 μm) were stained with periodic acid Schiff's (PAS) for neutral mucins.\n\nStatistics\nGene expression and body weight data were compared by ANOVA with a post-hoc Tukey's analysis (Systat software, Chicago, IL). Survival data were analyzed by a log-rank test for P values (PEPI software, ). The distributions of genotypes of pups surviving to weaning from breeding Cftr(+/-) mice were compared to that expected by Mendelian genetics using Chi-square analysis. For all statistical tests, P \u003c 0.05 was considered significant."}

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One group was congenic on the C57Bl/6 (B6) background, as previously described [21]. The other group was on a mixed background of about 95% B6 and 5% 129/Sv (129). The mice on the mixed background originated as part of a recently published study [20] as follows. Mice carrying a targeted mutation of the gastrin gene on a mixed B6/129 background [51] were bred for eight generations onto the B6 background. The gastrin(+/-) mice were then crossed for six generations with Cftr(+/-) mice congenic on the B6 background. A genome scan at this point showed that the mice were about 95% B6 and 5% 129 (see below). These mice were bred to obtain mice wild type for both gastrin alleles and either Cftr homozygous wild type [Cftr(+/+)] or Cftr homozygous null [Cftr(-/-)].\nMice were genotyped at 2 weeks of age by PCR as previously described [21]. Unless otherwise stated, mice were maintained on a complete elemental liquid diet (Peptamen; Nestle Deerfield, IL) to prevent intestinal obstruction that occurs in CF mice [52]. Wild type littermates were maintained on the same diet. In some experiments, 8 week old mice were transferred onto solid mouse chow instead of Peptamen, and survival was recorded. Mice with apparent distress were sacrificed and survival on chow was recorded as the following day. Male and female mice were used at 6–16 weeks of age. Mice were kept in a specific pathogen-free facility in barrier-top cages. All procedures were approved by the University of Kansas Medical Center IACUC.\n\nGenetic background determination\nThe Genome Scanning Service of The Jackson Laboratory (Bar Harbor, ME) was used to determine the contributions of C57Bl/6 and 129/Sv strains in the interbred mice. Pieces of mouse tail were sent to The Jackson Laboratory for simple sequence length polymorphism (SSLP) PCR analysis with the DMit primers specific for B6 and 129 strain alleles . The SSLP panel consists of 108 mapped markers designed to distinguish between B6 and 129 strains. The markers are spaced 12–13 cM apart and span the nineteen autosomes.\n\nMeasurement of gene expression\nTotal RNA was extracted from the entire small intestine as previously described [21]. Quantitative, real-time RT-PCR was used to measure expression of specific genes using the previously described primers [21]. Values were normalized to GAPDH mRNA and expression of this housekeeping gene is not altered in the CF mouse small intestine [21]. Expression of the major intestinal mucin, Muc2, was also measured using the forward (5'-GAC TTC GAT GGA CAC TGC TC-3') and reverse (5'-CAC GGT GTT TAT CTA CCA AC-3') primers.\n\nHistology\nThe small intestine was flushed with phosphate buffered saline and immersion fixed overnight in 4% paraformaldehyde. The tissues were then prepared for paraffin embedding and sectioning by a commercial service (HSRL, Woodstock, VA). Sections (5 μm) were stained with periodic acid Schiff's (PAS) for neutral mucins.\n\nStatistics\nGene expression and body weight data were compared by ANOVA with a post-hoc Tukey's analysis (Systat software, Chicago, IL). Survival data were analyzed by a log-rank test for P values (PEPI software, ). The distributions of genotypes of pups surviving to weaning from breeding Cftr(+/-) mice were compared to that expected by Mendelian genetics using Chi-square analysis. For all statistical tests, P \u003c 0.05 was considered significant."}