Figure 1: Validation of Noct WT mouse model.

A) Arrangement of the Wildtype (WT) Noct gene and TRE2 Noct transgene (pTRE2 is an expression plasmid with a tetracycline response element) containing the Tet-operator sequences, Noct open reading frame, and 3xFlag tag (not to scale). Primers used to amplify the Noct WT (P1), total Noct containing both WT and pTRE2 Flag (P2), and pTRE2 Noct 5’ untranslated region (P3) are indicated. pTRE2 (plasmid containing the tetracycline response elements), pCMV (cytomegalovirus promoter), TetO (Tetracycline operator), AatII and SapI (Restriction endonucleases used for cloning). B) Confirmation of Noct transgene expression in ear mesenchymal stem cells isolated from Noct WT animals and treated with 1 μg/mL Dox in vitro before fixation and staining for Flag expression. There is robust Flag expression (Green, FITC) showing Dox can induce NOCT-Flag protein expression. Nuclei stained with DAPI are in blue. C) Confirmation of Noct transgene expression in bone marrow stromal cells isolated from Noct WT animals and treated with 1 μg/mL Dox in vitro before western blotting for Flag expression. As can be seen, there is robust Flag expression, the same blot was probed with cyclophilin as a protein loading control. D)Total tibial bone RNA from 16 weeks old Noct WT mice showing expression of Noct using qRT-PCR and primers described in A (12 weeks on Sacch or Dox respectively). Values represent mean ± SEM of n ≥ 3 for qRT-PCR analysis. p values are calculated using student’s t-test. (NTC in fig 1B and 1C, represents no treatment control)