Fig 1: PR inactivation in the Mx1+ calvarial cells and calvariae in vitro.(A) A schematic diagram showing that PR-flox is crossed to Mx1-Cre. Mx1-Cre can be activated by IFNa to delete exon 2 of the PR gene to generate PR mutants (?PR). (B) Mx1-Cre;PR-flox/flox calvarial cells were treated with IFNa (500 units/mL) or without IFNa (control) for three days. The cells were then differentiated into osteoblasts in osteogenic medium without IFNa for 14 days. The relative expressions of RUNX2, Osteocalcin (Ocn) and DMP1 were evaluated by real-time PCR at day 14 and normalized to endogenous ß-actin. (C) The cells were collected for alkaline phosphatase (ALP) activity assays on day 10 and alizarin red staining (AR) on day 21. The optical density (OD) values were normalized to the corresponding total protein concentrations. Calvarial cells from the PR-flox/flox (without Cre) mice were used as a negative control to exclude the effect of INFa itself. (D) The calvariae obtained from Mx1-Cre;mT/mG double transgenic pups exhibited significant numbers (~40%) of cells that became GFP-positive after three days of IFNa (500 units/mL) treatment, and significantly more cells (~80%) became GFP-positive after an additional six days of culture without IFNa. (E) Genomic DNA was isolated from PR-flox/flox calvarial cells three days after IFNa treatment, and subjected to PR allele-specific PCR. The deleted PR band (?PR) indicated Cre-mediated DNA recombination. (F) Five-weeks-old PR-flox/flox or Mx1-Cre;PR-flox/flox mice were injected with pI-pC. Calvariae were collected five months later for microCT analysis for bone volume and (G) representative calvarial images from microCT scans.