This protocol can be used to generate a non-random mouse model for testing and quantifying the reactivation of the inactive X-linked Mecp2 chromosome in the brain of a living mouse. This model can be easily modified to study Xi reactivation in other X-linked disease models such as DDX3X syndrome. I am a post doc in Sanchita Bhatnagar's laboratory and I will be demonstrating the procedure with Zeming Zheng.
Begin by positioning the anesthetized mouse in a stereotactic platform with the incisors hooked in the bite bar of the snout restrainer. Tighten the nose clamp while keeping the mouse's head on a level plane. Adjust the height of the ear bars to reach the caudal portion of the ear canal to keep the head immobilized.
Then disinfect the head with alternating wipes of a topical antiseptic and 70%ethanol. When the mouse is ready, use a sterile scalpel to make a 0.75 centimeter horizontal incision in the mid scalp. Use a 0.45 millimeter burr to drill two symmetrical holes above the right and left cortical hemispheres two millimeters from the sagittal suture and the lambdoid suture at the approximate middle of the parietal bone.
Firmly attach a 10 microliter syringe to the stereotactic platform and load 10 microliters of freshly prepared chemical inhibitor into the syringe. Advance the needle into the first burr hole while keeping the needle perpendicular to the skull. When the needle traverses the skull, zero out the coordinates on the stereotactic digital display and advance the tip of the needle until it reaches a depth of 2.5 millimeters.
Withdraw the needle 0.5 millimeters to a depth of two millimeters and slowly inject the entire 10 microliter volume of the solution over a period of one minute. After all of the inhibitor has been delivered, leave the needle in the brain for another minute before withdrawing the needle. Repeat the injection into the second burr hole using vehicle only as a control and close the skin over the incision.
Then transfer the mouse from the stereotactic apparatus onto a 37 degree Celsius heating pad with monitoring until full recovery. At the end of the dose regimen, secure the mouse on a dissecting pad. Use scissors and forceps to make a lateral incision through the integument and the abdominal wall just beneath the ribcage.
Carefully separate the liver from the diaphragm and cut through the diaphragm along the entire length of the ribcage to expose the pleural cavity. Use the scissors to make an incision to the posterior end of the left ventricle and immediately inject the right heart chamber with approximately 15 milliliters of PBS over a two-minute period. A successful perfusion will result in a liver color change from red to pale pink.
When all of the PBS has been delivered, perfuse the heart with approximately 10 milliliters of 4%paraformaldehyde in PBS over two minutes. After harvesting the head, use scissors to make a midline incision in the scalp to expose the skull. Place one tip of the scissors into the foramen magnum to facilitate the creation of a lateral incision in the skull toward the eye.
Make a similar incision on the other side of the skull keeping the end of the scissors as superficial as possible and cut the region of the skull between the eyes and above the nose of the mouse. Use forceps to gently peel the cranial bones from the brain hemispheres and use a spatula to elevate the brain. Carefully dissect the cranial nerve fibers that fix the brain to the skull and place the brain into a plastic dish.
Then excise the cerebellum and olfactory bulbs. To obtain sections of the brain tissue, first fix the brain in a 15 milliliter tube filled with 4%paraformaldehyde in PBS at four degrees Celsius overnight. The next morning, rinse the brain tissue at least three times for five minutes at four degrees Celsius in fresh PBS per wash.
After the last wash, remove the front of each hemisphere with scissors and forceps and place the samples into labeled cryo-molds front side down. Submerge each tissue in optimal cutting temperature medium before snap freezing the samples in liquid nitrogen in a 10 millimeter plastic Petri dish for minus 80 degrees Celsius storage. To determine Xi reactivation, immerse five to six micrometer brain tissue sections in antigen retrieval solution for five minutes on a 100 degree Celsius heat block.
At the end of the incubation, wash the slides with four five-minute washes at room temperature with fresh PBS per wash before immersing the slides in blocking solution. After 20 minutes at room temperature, wash the slides three times for five minutes in fresh wash buffer per wash. Stain the tissue samples with the appropriate primary antibodies at four degrees Celsius overnight.
Image the slides on a fluorescence microscope adjusting the contrast and brightness to allow quantification of the number of GFP positive cells for both drug and vehicle treated mouse brain hemispheres. To demonstrate the feasibility of the non-random X chromosome inactivation mouse model for inactive X chromosome reactivation studies, the genotypes of female transgenic mouse embryonic fibroblast were confirmed by genotyping PCR and flow cytometry. As assessed by PCR, the expression of the GFP tagged methylene CPG binding protein two gene or MECP2 was reactivated by drug but not vehicle treatment.
Non-random X chromosome inactivation mouse model embryonic fibroblasts also expressed nuclear GFP after drug but not vehicle treatment demonstrating the X chromosome inactivation factor inhibitors reactivate inactivated X chromosome linked MECP2 in mouse embryonic fibroblasts. Drug treatment reactivates inactivated X chromosome MECP2-GFP in about 30%of cells in drug infused brain hemispheres whereas no reactivation is detected in vehicle infused hemispheres. Microtubule-associated protein two positive neurons are also GFP positive in drug treated hemispheres indicative of inactivated X chromosome MECP2-GFP expression.
The drug regimen will be dependent on the target and the efficacy of the small molecule inhibitors so optimize the dose and treatment in vitro before testing in vivo. The non-random mouse model can be utilized to test different drugs either individually or in combination. In fact, we and others have identified several druggable X chromosome inactivation factors.
