Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

Takako I. Jones; Guo-Liang Chew; Pamela Barraza-Flores; Spencer Schreier; Monique Ramirez; Ryan D. Wuebbles; Dean J. Burkin; Robert K. Bradley; Peter L. Jones

Stock TMX aliquots were warmed to 37°C, diluted with pre-warmed sterile corn oil either 10-or 20-fold just prior to use, and administered to mice by intraperitoneal (IP) injection at a final concentration of 5 or 10mg/kg. For the moderate model, ACTA1-MCM;FLExD bi-transgenic animals were injected once with 5mg/kg TMX. For the severe model, ACTA1-MCM;FLExD bitransgenic mice were injected on two consecutive days with 10mg/kg TMX. glutaraldehyde, 5mM EGTA, 100mM MgCl2 in PBS pH7.4) at 4°C for 4 hours, cryopreserved in cold 30% sucrose/PBS at 4°C overnight, then frozen in O.C.T. for sectioning. Cryosections (8µm) were fixed with 4% paraformaldehyde (PFA)/PBS, then stained with X-Gal staining solution (1X PBS with 2mM MgCl2, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 0.01% Nonidet P-40, 0.1% sodium deoxycholate, 1mg/mL X-Gal) for 50 min. Stained sections were post-fixed in buffered formalin at 4ºC for 5 min, rinsed three times with PBS and then deionized water before mounting. Images were captured using an Olympus CX41 microscope with PixeLINK camera under bright field.