1. Intestinal Vitamin D Receptor Is Required for Normal Calcium and Bone Metabolism in Mice 
Yingben Xue, James C. Fleet 
Gastroenterology 
Volume 136, Issue 4, Pages e2 (April 2009)
DOI: /j.gastro
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2. Figure 1
Generation of intestine-specific, hVDR-expressing, TG mice. (A) Transgene construct. A 12.4-kb villin promoter/enhancer was used to drive expression of a HA-tagged–hVDR cDNA transgene in the intestine. Primers P1 and P2 were used for genotyping; primers P3 and P2 were used for assessing transgene mRNA levels. (B) Genotyping of transgenic offspring yields a 347-bp PCR product. w, WT mice; t, transgenic mice. (C) Transgene mRNA expression in intestinal segments of WT and TG(H) mice (dd, duodenum; je, jejunum; il, ileum; ce, cecum; cop, proximal colon; cod, distal colon). Transgene mRNA, 392-bp PCR product. (D) Transgene mRNA expression in a survey of mouse tissues.
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
Characterization of transgene mRNA and protein levels in 2 transgenic lines. TG and total VDR mRNA levels in (A) duodenum and (B) kidney of WT, TG(L), and TG(H) mouse lines were determined. Bars represent the mean ± SEM (n = 4). *P < .05 compared with WT, +P < .05 compared with TG(L). (C) Total VDR protein levels in duodenum and (D) in different segments of intestine in WT and TG(H) mice were determined using a rat anti-VDR antibody by Western blot analysis as described in the Materials and Methods section. The band density values were measured by Image J software (National Institutes of Health, Bethesda, MD; available: and are shown on the Western blot image relative to the values for the WT duodenum. (vdr std, VDR protein standard).
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
Gross appearance, growth curves, and serum chemistries of VDR+/-, KO, and KO/TG mice. (A) Physical appearance and (B) growth curves of 12-week-old male heterozygotes (VDR+/-), VDR KO, and KO mice recovered with low or high expressing transgene, KO/TG(L) or KO/TG(H), respectively. Symbols represent the mean ± SEM (n = 8). *P < .05 compared with VDR+/- mice. (C) Serum Ca (a) and PTH (b) levels. Each value is the mean ± SEM (n = 8 per genotype). *P < .05 compared with VDR+/- mice. (c) Representative H&E-stained histologic sections of parathyroid glands (arrows) and adjacent thyroid tissue. Original magnification, 10×; scale bar = 254 μm.
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
Intestinal phenotype of VDR+/-, KO, and KO/TG mice. (A) Duodenal Ca absorption (a), CYP24 (b), calbindin D9k (CaBP9K), and TRPV6 (c) mRNA levels in 8-week-old female VDR+/-, KO, and KO/TG(H) mice 9 hours after treatment with 25 ng or 100 ng of 1,25(OH)2D/100 g body weight or vehicle (control). Bars represent the mean ± SEM (n = 6–8). (B) CaBP9K and TRPV6 mRNA levels in the proximal colon of 12-week-old male VDR+/-, KO, and KO/TG(H) mice (a) and in 8-week-old female VDR+/-, KO, and KO/TG(H) mice (b) 9 hours after treatment with 25 ng of 1,25(OH)2D/100 g body weight or vehicle (control). Each value is the mean ± SEM (n = 6–8 per genotype). *P < .05 compared with control-treated VDR+/-, #P < .05 compared with 1,25(OH)2D-treated VDR+/- mice, +P < .05 compared with control-treated KO/TG(H), $P < .05 compared with 25 ng/100 g body weight (bw) 1,25(OH)2D-treated KO/TG(H). mRNA levels were assessed by real-time PCR analysis, normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level, and expressed relative to levels of VDR+/- mice.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Renal phenotypes of VDR+/-, KO, and KO/TG mice. (A) Twelve-week-old male VDR+/-, KO, KO/TG(L), and KO/TG(H) were examined for basal levels of the following: (a) serum 1,25(OH)2D, (b) renal CYP27B1 and CYP24 mRNA levels, (c) urine Ca/creatinine ratio, and (d) renal calbindin D9k (CaBP9K) and calbindin D28k (CaBP28K) mRNA levels. Each value is the mean ± SEM (n = 8 per genotype). *P < .05 compared with VDR+/- mice, #P < .05 compared with KO mice, +P < .05 compared with KO/TG(L) mice. (B) Eight-week-old female VDR+/-, KO, and KO/TG(H) mice treated with 25 ng of 1,25(OH)2D/100 g body weight or vehicle (control) for 9 hours and renal RNA was examined for the following: (a) CYP24, (b) CYP27B1, and (c) TRPV5 mRNA levels by real-time reverse-transcription PCR analysis. Values were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels and are expressed relative to levels in control-treated VDR+/- mice. Bars represent the mean ± SEM (n = 6–8). *P < .05 compared with control-treated VDR+/- mice, #P < .05 compared with 1,25(OH)2D-treated VDR+/- mice, +P < .05 compared with control-treated KO/TG(H).
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Figure 6
Skeletal phenotype of VDR+/-, KO, KO/TG(L), and KO/TG(H) mice. (A) Femoral BMD of 12-week-old male of VDR+/-, KO, KO/TG(L), and KO/TG(H) was determined using dual-energy x-ray absorption. Bars represent the mean ± SEM (n = 8). *P < .05 compared with VDR+/- mice. (B) Representative contact radiographs of the femurs. Arrows are pointing to the epiphysis, metaphysis, and diaphysis (left to right).
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8. Figure 7
Static and dynamic histomorphometric analysis of VDR+/-, KO, and KO/TG(H) mice. Decalcified paraffin-embedded femoral sections from 12-week-old male VDR+/-, KO, and KO/TG(H) were examined for (A and F, a) trabecular bone volume (trabecular bone volume/total tissue volume) by total collagen staining; (D and F, d) osteoblast number (cell number/mm2 of tissue area) from H&E-stained sections; and (E and F, e) osteoclast number (cell number/mm2 of tissue area) from tartrate-resistant acid phosphatase (trap) staining. Calcified, plastic-embedded, femoral sections from 8-week-old female VDR+/-, KO, and KO/TG(H) mice treated twice with calcein were used for (B and F, b) mineralization (osteoid volume/total bone volume [ov/bv]) from von Kossa staining, and (C and F, c) mineral apposition rate (mar). (A–E, left column) Representative micrographs of histologic sections are shown. The original magnification in these sections is: 4×, 40×, 20×, 60×, and 40×, respectively, and the scale bars represent 254 μm, 127 μm, 100 μm, 127 μm, and 127 μm, respectively. Arrows point to the following: (A) growth plate, (B) osteoid, (C) calcein labeling, (D) osteoblast, and (E) osteoclast. (F) Quantification of these data is presented in the graphs. Bars represent the mean ± SEM (n = 8). *P < .05 compared with VDR+/- mice. #P < .05 compared with KO mice.
Gastroenterology  , e2DOI: ( /j.gastro )
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