Td96348

The Vdr-KO line was generated in the Demay lab and bred in house at MGH for several years to ensure reproducibility of results when compared to similarly bred WT mice. To normalize the blood mineral ion levels in Vdr-KO−/− mice, these animals were fed a γ-irradiated rescue chow (TD96348, Teklad, Madison, WI) containing 2% calcium, 1.25% phosphorus, and 20% lactose with 2.2 IU vitamin D/g (Li et al., 1998 (link)). Feces were collected from mice that were ~8 weeks of age, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis.

VDR-null and Cyp27b1-null mice and transgenic strains were maintained as heterozygotes through outbreeding with C57BL/6 mice (The Jackson Laboratory). All mice were fed standard rodent chow diet (5008; Harlan Teklad) after weaning except VDR-null mice to examine alopecic phenotype at 6 month of age. In this case, the mice were fed on a diet containing 20% lactose, 2% calcium and 1.25% phosphate diet (TD.96348; Harlan Teklad). For gene expression analysis, mice were treated with 1,25(OH)₂D₃ (10 ng/g body weight, SAFC) for 6 h, atRA (1 ng/g body weight, Sigma, R2625) for 4 h, fibroblast growth factor 23 (FGF23, 50 ng/g body weight, R & D Systems, 2629-FG-025) for 3 h or PTH (230 ng/g body weight, Bachem, H-1370.0100) and dibutyryl cyclic AMP (db-cAMP, 0.1 ng/g body weight, Sigma, D0260) for 1 h. For ChIP-seq analysis, mice were treated with 1,25(OH)₂D₃ (10 ng/g body weight) for 1 h. Mouse treatments were performed by intraperitoneal injection. The mice sacrificed for gene expression and ChIP-seq analyses were 8–10 weeks old of both genders. Bone mineral density (BMD) was measured using 8 weeks old male mice. Images of mouse gross appearance to check the presence of alopecia was taken at 6 month of age. Mice were exposed to a 12-h light-dark cycle. All animal studies were reviewed and approved by the Research Animal Care and Use Committee of University of Wisconsin-Madison.