C57bl 6 mouse

All animal experiments were approved by the Peking University Animal Care and Use Committee. All methods were performed in accordance with the relevant guidelines and regulations. Human DLX3 mutation (Q178R) gene flanked by the mouse cytokeratin 14 promoter was subcloned into plasmid pGL647. The fragments of the DLX3 mutation (Q178R) transgene were purified and microinjected into C57BL/6 mouse oocytes, and the oocytes were surgically transferred to pseudopregnant C57BL/6 mouse (Cyagen Biosciences Inc., Guangzhou, China). Initial genotyping of founder transgenic mice was performed by PCR using tail biopsies from pups. Positive founders for transgene were reconfirmed by western blot analysis to test the overexpression of DLX3. Transgenic mice were bred with wild type mice to generate (to obtain a defined genetic background) hemizygotes for the analysis of bone phenotype changes. In all experiments, female transgenic mice and female wild type littermates as controls were used. We established a sample of at least 5 mice per group.

MSCs derived from bone marrow of female C57BL/6 mouse were purchased from Cyagen Bioscience, Inc. (Guangzhou, China) and general identified according to surface phenotypes and multipotency by supplier. DMEM/F12 containing 10% fetal bovine serum growth medium (Wisent, Nanjing, China) and humidified 5% CO2 incubator at 37 °C were suitable for MSC cultivate. And culture media were changed every 2–3 days. MSCs of passages 3–7 from the same batch were finally used for all experiment.
MSCs were cultured and divided into the normoxia culture group (20% O2) and hypoxia culture group (1% O2) through inflating different nitrogen. Each group received different treatments for 0 to 24 h. Before some experiments, MSC were treated with HIF-1α inhibitor KC7F2 (20 μM, Selleck, USA). For Transwell migration assay, SDF-1 (100 ng/ml, Millipore, MA) were used.