SEPN1-related myopathy depends on the ER oxidase ERO1 alpha and is responsive to the chemical chaperone TUDCA
SEPN1-related myopathy depends on the ER oxidase ERO1 alpha and is responsive to the chemical chaperone TUDCA
Age Males WT-Sepn1 Ko-Chop Ko-Sepn1 Ko/Chop Ko OLD: 1 year and 6 months (18months). Age Males WT-Sepn1 Ko-Chop Ko-Sepn1 Ko/Chop Ko YOUNG: 2 months. Age Males WT-Sepn1 Ko-Ero1 Ko-Sepn1 Ko/Ero1 Ko YOUNG: 9 months. The SELENON KO mice were purchased from the EMMA repository (SELENON<tm1.2Mred>/Orl). The CHOP KO mice were purchased (Stock No. 005530) from Jackson Laboratory. The ERO1 alpha KO mice line was resuscitated from the embryos of David Ron's stock [(Chin, K. T., et al. (2011). "The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load." FASEB J 25(8): 2583-2591]. Fill the becher with isopentane and put it in dry ice in order to reach the correct temperature. Sacrifice the mice by dislocation. Fix the mouse in supine position on the surgical mat by using the needles. Using surgical scissors and forceps, cut the mouse skin to expose the peritoneum. Once that the area is cleaned from hairs, use scissors and forceps to cut the peritoneum. Now that the rib cage and internal organs are exposed, it is necessary to remove everything that is close to the diaphragm (liver, gallbladder, etc). Once that the diaphragm is clearly visible, tilt the rib cage upward using the forceps and cut the diaphragm following the rib cage with the scissors (with a circular movement). Once that the diaphragm is detached from the rib cage, clean it of any residuals by using the forceps. Wash the diaphragm in PBS to remove the blood. Put the diaphragm in cold isopentane in order to instantly freeze the muscle. Store the samples at -80°C.  Collection of 5-10 million cells in PDS, and resuspension of the pellet in a buffer. Crumble 50/60mg of muscles by using liquid nitrogen and a pestle. Once that the muscles is mechanically lisate, add 1mL of QIAzol® lysis reagent (by QIAGEN) and put it at -80°C overnight. Extract the RNA the Qiagen RNeasy kit. TruSeq Stranded Total RNA Sample Preparation Guide