Fig. 7. Targeted disruption of the Organic Solute Transporter alpha subunit gene (Osta). (A) Schematic representation of the wild-type Osta gene (Wild type Osta allele) showing the locations of exons 1 to 9 and the SacI restriction enzyme cleavage sites used to construct the conventional targeting vector. The murine Osta gene is organized in 9 exons spanning »13-kb of DNA sequence. The first exon encompasses the 5' untranslated region and encodes amino acids 1 to 13. Exons 2 through 9 include the remaining coding sequence (amino acids 14 to 340). Exon 9 also encodes a 3' untranslated region of 205 nucleotides. Exons 1, 2, and the proximal promoter region are replaced by the neomycin resistance gene (neo) as a result of homologous recombination (Targeted Osta allele), such that the neomycin gene is transcribed in the opposite direction of the Osta gene. After deletion of these coding exons, the next potential in-frame methionine lies in exon 3 at position 117. In the event that an aberrant Osta transcript is generated from the targeted locus, the primary translation product would be a mutant protein lacking the first 116 aa, including the extracellular amino terminus and the first 2 potential transmembrane domains. (B) Southern blot analysis of HindIII-BamHI (left panel) or HindIII-SspI (Right) digested genomic DNA from wild-type embryonic stem cells (WT) or the embryonic stem cell clone (2C3) that was injected into blastocysts to produce the Osta^-/-mice. The blots were hybridized with a [a-³²P]dCTP-labeled 361 bp StuI-XbaI fragment (Probe A) derived from Osta 5' flanking sequence immediately upstream of the short arm. (C) Genotype analysis of mice with different Osta alleles. Schematic representation of the wild-type and targeted Osta gene showing the locations of allele-specific PCR primers used for genotype analysis. Genomic DNA was isolated from animals of the indicated genotype and PCR amplified using the indicated primers as described in Materials and Methods. The products were resolved on a 1% agarose gel and their sizes are indicated.

Fig. 8. Expression of Osta-Ostbm RNA and protein in Osta^-/-mice. (A) Northern blot analysis of Osta and Ostb mRNA in wild-type and Osta^-/-mice. The small intestines were isolated from wild-type and Osta^-/-male mice (n = 5 mice per genotype; 3 months of age; mixed C57BL/6J-129/SvEv background), divided into five segments of equal length, and used to isolate RNA. Pooled aliquots of total RNA (10 mg) were subjected to Northern blot hybridization using radiolabeled full-length cDNA probes for mouse Osta, Ostb, and b-actin. No Osta mRNA was detectable in small intestine from the Osta^-/-mice, indicating that replacement of the 5' region of the Osta gene with the neomycin resistance cassette region abolished transcription of the Osta gene or generated an unstable transcript that is rapidly degraded. In contrast, Ostb mRNA remained highly expressed in small intestine of the Osta^-/-mice. (B) Ileal expression of Ostaand Ostb protein in wild-type and Osta^-/-mice. The small intestines were isolated from male and female mice of the indicated genotypes (n = 2 mice per group, 4 months of age; mixed C57BL/6J-129/SvEv background), divided into five segments of equal length, and terminal ileum (segment 5) was used to prepare tissue protein extracts. Equivalent amounts (50 mg) of ileal protein extract from the indicated genotypes were resolved by SDS/PAGE, transferred to nitrocellulose, and probed with anti-Osta or anti-Ostb antibody. The blots were then reprobed using an anti-b-actin antibody as a control for protein loading.

Fig. 9. Body weights of wild-type, Osta^+/-, and Osta ^-/-mice. Mean values ± SE are shown. (A) Males (n = 7-18 mice per genotype). (B) Females (n = 7-15 mice per genotype). An asterisk indicates body weights of Osta^-/-mice that are significantly different from wild type and heterozygous mice (*P < 0.05).

Fig. 10. Taurocholate transport in everted gut sacs. (A) Small intestines from Osta^+/+ male mice were divided into four equal parts, everted, and used to prepare gut sacs. The individual everted sacs were incubated for 30 min at 37°C in oxygenated KRB containing 25 mM [³H]taurocholate and inulin [¹⁴C]carboxylic acid (as a control for leakage and paracellular movement). Mean values ± SE are shown (n = 3-5 mice per segment). Taurocholate transport was largely restricted to the distal portion (ileum) of the small intestine. (B) Taurocholate content of tissue extracts prepared from jejunal and ileal gut sacs. After rinsing the sacs and removing the serosal fluid, the gut sacs were solubilized and used to determine the amount of tissue-associated taurocholate. Mean values ± SE are shown (males, n = 3-6 mice per segment; females, n = 3-7 mice per segment). Different letters represent significant differences between groups (P < 0.05 by Tukey's honestly significant difference test). The tissue-associated taurocholate was significantly lower in sacs prepared from the Osta^-/-mice versus wild-type mice (Males and Females). (C) Efflux of taurocholate from the tissue to the serosal side. The amount of taurocholate measured in serosal fluid (Fig. 2) was divided by the amount of taurocholate associated with the tissue (Fig. 10B).

Fig. 11. Fecal bile acid excretion in wild-type and Osta^-/-mice. Fecal bile acid excretion was measured in mice at 1 month of age as described in Materials and Methods. Mean ± SE are shown (n = 10 mice per group). At 1 month of age, fecal bile acid excretion levels were similar in the wild-type and Osta^-/-mice.