The authors thank Antonio Bedalov for providing reagents; University of Virginia Tissue Histology Core for cryosectioning; University of Virginia Flow Cytometry Core for flow cytometry analysis; Christian Blue and Saloni Singh for technical assistance with genotyping. This work was supported by a Double Hoo Research Grant to Z.Z., and a Pilot Project Program Award from the University of Virginia-Virginia Tech Seed Fund Award and the Hartwell Foundation Individual Biomedical Research Award to S.B.

Work involving mice was approved by the University of Virginia Institutional Animal Care and Use Committee (IACUC; #4112).
1. Generate a Non-random XCI Mouse Model with Genetically Labeled Mecp2 on Xi
NOTE: Mouse strains used in the study were as follows: Mecp2-Gfp/Mecp2-Gfp (Mecp2tm3.1Bird, Table of Materials) and Xist/&#916;Xist (B6;129-Xist&#60;tm5Sado&#62;; provided by Antonio Bedalov, Fred Hutchinson Cancer Center, Seattle). Breeding strategies among the respective strains have been designed to expand the mouse colonies for each strain.
<ol>
	<li>Perform PCR-genotyping on all mice strains and respective progenies obtained after breeding using gene specific primers listed in Table 1.</li>
</ol>
2. Design the Mouse Breeding Strategy to Generate Xist&#916;:Mecp2/Xist:Mecp2-Gfp
<ol>
	<li>Set up a breeding pair by housing a Mecp2-Gfp/Y male and a Xist&#916;-Mecp2/Xist-Mecp2 female together (12 h/12 h: light / dark cycle)&#160;&#160;(Figure 1A). Ideally, set up at least 5 breeding pairs at a time using viable and fertile mice. After the pregnant female gives birth, allow her to raise her first litter with the male. 
	NOTE: Because paternal Xist knockout impairs imprinted XCI, dosage compensation, and differentiation pathways17, the use of Xist&#916;-Mecp2/Y mice in the breeding will fail to produce the female pups with required genotype.</li>
	<li>After weaning the litter at post-natal day 21 (P21), identify and tag the Xist&#916;:Mecp2/Xist:Mecp2-Gfp female pups using a PCR-based genotyping assay (Figure 1B). 
	NOTE: In terms of the number of animals needed per group, for the results reported below, 5 breeding pairs were set up which generated approximately 10 female Xist&#916;:Mecp2/Xist:Mecp2-Gfp mice.</li>
	<li>Use female mouse models for all the proposed experiments. 
	NOTE: Sex is an important biological variable for XCI studies, and the male model does not account for the confounding effects of random XCI.</li>
	<li>To be consistent throughout the animal studies and rule out any effects of the animal age, perform all the experiments in 5&#8722;8-week-old female Xist&#916;:Mecp2/Xist:Mecp2-Gfp mice.</li>
</ol>
3. Isolate Female Xist&#916;:Mecp2/Xist:Mecp2-Gfp Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Set up timed mating between the Mecp2-Gfp/Y male and Xist&#916;-Mecp2/Xist-Mecp2 female. Set up at least 3&#8722;4 mating cages to increase the likelihood of pregnancy.</li>
	<li>After confirming the vaginal plug the morning after mating, separate the female and this day is considered embryonic day 0.5 (E0.5). Monitor the weight gain of the prospective pregnant female and visually inspect the abdomen of the mice to confirm pregnancy. On E14.5&#8722;15.5, euthanize the pregnant female mice via cervical dislocation.</li>
	<li>Under a laminar hood, wipe the abdomen of the pregnant female with 70% ethanol. Using sterile scissors, dissect the abdominal cavity and remove the uterine horns containing the embryos. Using forceps, gently take out the embryos while cutting off the remaining abdominal tissue (6&#8722;12 embryos is usually expected).</li>
	<li>Place the uterine horns containing the embryos in a 10 mm tissue-culture dish. Using sterile scissors and forceps, make an incision along the uterine horns to isolate individual sacs carrying an embryo. Carefully, place the uterine horns containing the rest of the embryos in 30 mL of sterile Hanks&#8217; balanced salt solution (HBSS) on ice.</li>
	<li>Gently cut open the sac and isolate the embryo using sterile scissors and forceps. Remove the placenta by cutting the umbilical cord.</li>
	<li>Decapitate the embryo. 
	NOTE: While the rest of the embryonic tissue will be further processed, the tissue from the embryo head will be used to isolate DNA for genotyping.</li>
	<li>Open the abdomen by cutting the midline of the embryo using sterile scissors and forceps. Remove the visceral organs, such as the heart, liver, and lungs.</li>
	<li>Transfer the remaining embryonic tissue into a sterile 60 mm tissue-culture plate and cut into small pieces using scissors or a razor blade. To break open the cell clumps, add 3 mL of trypsin-EDTA (0.05%) and incubate at 37 &#176;C for 15 min.</li>
	<li>Neutralize trypsin-EDTA by adding 5 mL of Dulbecco&#8217;s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 10 &#181;g/mL penicillin/streptomycin (pen/strep) to the plate and dissociate the tissue by repetitive pipetting (approximately 20&#8722;30 times).</li>
	<li>Spin down cells at 300 x g for 5 min and re-suspend the cell pellet in 4 mL of DMEM with 10% FBS and 10 &#181;g/mL pen/strep. Plate the cells on a 60 mm culture dish, and culture the cells at 37 &#176;C in the presence of 5% CO2.</li>
	<li>Carry out steps 3.5&#8722;3.10 for each embryo.</li>
	<li>Culture MEFs obtained in step 3.11 for at least 3&#8722;4 days. 
	NOTE: At this stage, MEFs can also be cryopreserved for future experiments.</li>
	<li>To determine the sex and genotype of each embryo, carry out genotyping-PCR using DNA isolated from the heads of the embryos using primers and PCR conditions listed in Table 1.</li>
</ol>
4. Confirm the Lack of Green Fluorescent Protein (GFP) Expression in the Brain of Xist&#916;:Mecp2/Xist:Mecp2-Gfp Mice Using a Ffluorescence Activated Cell Sorting-based Assay
<ol>
	<li>Using scissors, separate the cerebral cortex from the rest of the brain hemispheres, and place in 1 mL of ice-cold nuclei isolation media (NIM) buffer containing 250 mM sucrose, 25 mM potassium chloride (KCl), 5 mM magnesium chloride (MgCl2), 10 mM Tris-Cl, supplemented with 2% paraformaldehyde (PFA), and 0.1% nonionic surfactant (Table of Materials).</li>
	<li>Homogenize using an ice-cold glass homogenizer.</li>
	<li>Spin down homogenized tissue at 600 x g, 4 &#176;C for 5 min.</li>
	<li>Remove supernatant and resuspend pellet in 1 mL of 25% iodixanol in NIM solution.</li>
	<li>Add 1 mL of 29% lodixanol in NIM solution to a 4 mL ultracentrifuge tube (store on ice until samples are ready) and carefully layer 1 mL of sample (in 25% lodixanol).</li>
	<li>Centrifuge at 9,000 x g, 4 &#176;C for 10 min in an ultracentrifuge.</li>
	<li>Aspirate supernatant and resuspend pellet in 500 &#956;L of fluorescence activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] supplemented with 1 mM EDTA, 0.05% sodium azide, and 2% bovine serum albumin [BSA]). 
	NOTE: Samples can be stored at 4 &#176;C or up to 1 week.</li>
	<li>Add 5 &#956;L of 7-amino-actinomycin D (7-AAD; 50 mg/mL) for 5 min at room temperature to stain DNA.</li>
	<li>Analyze samples for 7-AAD and green fluorescent protein (GFP) signal using flow cytometry.</li>
</ol>
5. Determine Feasibility of the Xist&#916;:Mecp2/Xist:Mecp2-Gfp Mouse Model for Xi Reactivation
<ol>
	<li>Seed 1 x 105 cells/mL female Xist&#916;:Mecp2/Xist:Mecp2-Gfp MEFs, Mecp2/Mecp2-Gfp and Xist-Mecp2/Y MEFs obtained in step 3.12 in a 6-well format and in chamber slides, in DMEM with 10% FBS and 10 &#181;g/mL pen/strep, at 37 &#176;C in the presence of 5% CO2. 
	NOTE: Mecp2/Mecp2-Gfp and Xist-Mecp2/Y MEFs are used as positive and negative controls in the experiments.</li>
	<li>Add fresh medium to the cells after 24 h. For Xist&#916;:Mecp2/Xist:Mecp2-Gfp MEFs, add medium supplemented with XCIFs inhibitors (e.g., 0.5 &#956;M LDN193189 and 2.5 &#956;M GSK650394), or vehicle alone. Replace medium supplemented with fresh inhibitor or vehicle alone every 2 days. For Mecp2/Mecp2-Gfp and Xist-Mecp2/Y MEFs, add fresh medium every 2 days.</li>
	<li>Post 1 week of inhibitor treatment, harvest MEFs either for RNA isolation (6-well plate) or fix cells for immunofluorescence (chamber slides). Use MEFs isolated from Mecp2/Mecp2-Gfp embryos as positive and Xist-Mecp2/Y embryos as negative controls respectively.
	<ol>
		<li>For RNA isolation, isolate total RNA by guanidinium thiocyanate-based RNA extraction reagent, and reverse transcribe using reverse transcriptase. Measure Mecp2-Gfp expression by quantitative reverse transcriptase-PCR (qRT-PCR) using Mecp2-WT and Mecp2-GFP, and primers listed in Table 1, as described previously12.</li>
		<li>For immunofluorescence, stain MEFs with an anti-GFP primary antibody (1:100), as described previously12,16. Measure GFP intensity by quantitative immunofluorescence in drug treated Xist&#916;:Mecp2/Xist:Mecp2-Gfp MEFs, as described previously18.</li>
	</ol>
	</li>
</ol>
6. Demonstrate the Pharmacological Xi Reactivation in the Brain of the Xist&#916;:Mecp2/Xist:Mecp2-Gfp Mouse Model
<ol>
	<li>Prepare drugs and vehicle control.
	<ol>
		<li>Prepare a fresh, sterile solution of vehicle (0.9% NaCl, 0.5% methylcellulose, 4.5% dimethyl sulfoxide [DMSO]) for brain injections.</li>
		<li>Prepare chemical inhibitors including 1.5 mM LDN193189 (small molecule inhibitor of ACVR1) and 1.6 mM GSK650394 (small molecule inhibitor of SGK1, a downstream effector of PDPK1) re-suspended in vehicle (0.9% NaCl, 0.5% methylcellulose, 4.5% DMSO), or vehicle alone. The total volume of chemical inhibitors or vehicle injected is 10 &#181;L per dose per animal.</li>
	</ol>
	</li>
	<li>Prepare animal for brain injections.
	<ol>
		<li>Prepare the surgical area by wiping the bench and heating pad with disinfectant (10% sodium hypochlorite solution).</li>
		<li>Anesthetize the 4 week old mouse with an intraperitoneal injection of ketamine/xylazine mixture at a dose of 140 mg/kg and 10 mg/kg, respectively. Use the pedal withdrawal reflex to determine the level of anesthesia.</li>
		<li>Apply ophthalmic ointment to the eyes following induction of anesthesia to prevent corneal drying.</li>
		<li>Shave off the fur from the neck to the top of the head of the mouse.</li>
		<li>Position the mouse in the stereotactic platform by hooking the mouse&#8217;s incisor teeth in the bite bar of the snout restrainer and tightening the nose clamp over the snout while ensuring that the mouse&#8217;s head is on a level plane.</li>
		<li>Adjust the height of the ear bars, as necessary, to reach the caudal portion of the ear canal, securing them such that the mouse&#8217;s head is in a level plane and immobilized on finger touch.</li>
		<li>Disinfect the head of the mouse with alternating wipes of a topical antiseptic, such as povidone-iodine and 70% ethanol.</li>
	</ol>
	</li>
	<li>Administer drugs.
	<ol>
		<li>Using a sterile scalpel, make a 0.75 cm horizontal incision in the mid-scalp.</li>
		<li>Using a 0.45 mm burr, drill two symmetrical holes above the right and left cortical hemispheres (2 mm from the sagittal suture and 2 mm from the lambdoid suture, approximately the middle of parietal bone).</li>
		<li>Attach a 10 &#956;L syringe to the stereotactic platform firmly.</li>
		<li>Mix the solution of chemical inhibitors, and draw 10 &#956;L of solution into the syringe. Avoid any air bubbles in the syringe.</li>
		<li>Advance the syringe needle into the burr hole maintaining the needle perpendicular (90&#176;) to the skull. When the needle traverses the skull, zero out the coordinates on the stereotactic digital display and then advance the tip of the needle until it reaches a depth of 2.5 mm.</li>
		<li>Withdraw the needle 0.5 mm to the depth for 2 mm.</li>
		<li>Slowly inject 10 &#956;L of solution (~1 min). After injection is complete, leave the needle in the brain for ~1 min and then withdraw the needle.</li>
		<li>Repeat the injection for the second hemisphere (vehicle only control).</li>
		<li>Using sutures or &#8220;skin glue&#8221; close the skin of the mouse (if wound is &#62; 0.5cm both sutures and &#8220;skin glue&#8221; should be used).</li>
		<li>Loosen the ear bars and remove the mouse from the stereotactic apparatus.</li>
		<li>Administer analgesics (Buprenorphine SR 0.5 mg/kg IP) and place the mouse on a heating pad set to 37 &#176;C until the animal regains consciousness.</li>
		<li>Once the mouse is alert and responsive, transfer the animal back to its original cage.</li>
		<li>Repeat the procedure every 2 days for 20 days. Repeat drilling of the area is not required for subsequent injections.</li>
	</ol>
	</li>
	<li>Isolate the mouse brain.
	<ol>
		<li>Once the dose regimen is completed, euthanize the mouse in a CO2 chamber.</li>
		<li>Immobilize the mouse on a surface using needles.</li>
		<li>Make a lateral incision through the integument and abdominal wall just beneath the rib cage using scissors and forceps. Carefully separate the liver from the diaphragm.</li>
		<li>Using scissors, cut the diaphragm and continue cutting along the entire length of the rib cage to expose the pleural cavity.</li>
		<li>Using scissors, make an incision to the posterior end of the left ventricle.</li>
		<li>Immediately, start injecting the right heart chamber with ~15 mL of PBS over ~2 min. Liver color change from red to pale pink is indicative of good perfusion.</li>
		<li>Inject the right chamber of the mouse heart with ~10 mL of 4% paraformaldehyde in PBS over ~2 min.</li>
		<li>Decapitate the mouse, and use scissors to make a midline incision of the scalp to expose the skull.</li>
		<li>Place one tip of the scissors into the foramen magnum, and cut laterally into the skull toward the eye. Repeat for the other side. Try to keep the end of the scissors as superficial as possible to avoid injury of the brain.</li>
		<li>Use scissors to cut the region between the eyes and above the nose of the mouse.</li>
		<li>Use forceps to gently peel the cranial bones from the brain hemispheres.</li>
		<li>Lift the brain with a spatula, and use scissors to carefully dissect the cranial nerve fibers that fix it to the skull.</li>
		<li>Place the brain on a plastic dish, cut out the cerebellum and olfactory bulbs, and place the brain into a 15 mL tube filled with 4% PFA is PBS.</li>
	</ol>
	</li>
	<li>Cryo-section the mouse brain.
	<ol>
		<li>Fix brain in 4% paraformaldehyde in PBS at 4 &#176;C overnight.</li>
		<li>Rinse the brain with PBS at 4 &#176;C at least 3x for 5 min each.</li>
		<li>Label the disposable molds for cryopreserving the tissues.</li>
		<li>Cut out the front of the brain using scissors and forceps (start making ~5 mm sections from injection sites).</li>
		<li>Transfer the brain to the cryomold with the front of the brain facing down to obtain coronal sections. Submerge the mold containing brain in the optimal cutting temperature compound (OCT).</li>
		<li>Pour liquid nitrogen into a 10 mm plastic petri dish and place the brain in the cryo-mold into the nitrogen. 
		NOTE: It is important to orient the tissue as described in step 6.5.5 to guide the sectioning of the brain.</li>
		<li>When the OCT is solid white, place the frozen brain into an -80 &#176;C freezer for storage.</li>
		<li>Equilibrate the brain to -20 &#176;C for at least 30 min prior to sectioning with cryostat and cryosection (5&#8722;6 &#956;m thickness) the brain, mounting 2&#8722;3 sections per slide. Slides can be stored at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Determine Xi reactivation using an immunofluorescence-based approach.
	<ol>
		<li>Before starting the staining procedure, dry the brain sections overnight at 4 &#176;C.</li>
		<li>Immerse slides in antigen retrieval solution (0.1 M citric acid, 0.1 M Tris-base, pH = 6.0) on a 100 &#176;C heat block for 5 min.</li>
		<li>Wash slides 4x with 1x PBS for 5 min each at room temperature.</li>
		<li>Immerse slides in blocking solution (0.1 M NH<s>4</s>Cl/PBS/0.2% gelatin/0.05% nonionic surfactant) for 20 min at room temperature.</li>
		<li>Wash slides 3x with wash buffer (PBS/0.2% gelatin) at room temperature for 5 min each.</li>
		<li>Stain brain sections with anti-GFP-AlexaFluor647 (1:100) and anti-MAP2 (1:1,000) antibodies in incubation medium (PBS/0.2% gelatin/1% BSA) and incubate at 4 &#176;C overnight.</li>
		<li>Collect primary antibodies (can be reused). Wash slides 4x with wash buffer for a total of 30 min at room temperature.</li>
		<li>Incubate brain sections with goat anti-chicken Fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:1,000) in incubation medium and incubate 1&#8722;2 h at room temperature in the dark.</li>
		<li>Wash slides 4x with wash buffer for a total of 30 min at room temperature.</li>
		<li>Place a drop of mounting medium with 4&#8242;,6-diamidino-2-phenylindole (DAPI) on a 22 mm x 50 mm coverslip, then invert the coverslip onto a slide, covering all tissue sections.</li>
		<li>Image on a microscope and adjust images for contrast and brightness. Capture images and quantify the number of GFP-positive cells for both drug-treated and vehicle treated Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse brain hemispheres.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Previously, XCIFs that are selectively required for silencing of Xi-linked genes in mammalian female cells were identified12. We further optimized potent small molecule inhibitors to target XCIFs, such as ACVR1 and downstream effectors of PDPK1, which efficiently reactivate Xi-linked Mecp2 in mouse fibroblast cell lines, mouse cortical neurons, and a human fibroblast cell line derived from a RTT patient. These results suggest that Xi reactivation is a plausible therapeutic approach to res...

X chromosome inactivation (XCI) is a process of dosage compensation that balances X-linked gene expression by silencing one copy of the X chromosome in females1. As a result, the inactive X chromosome (Xi) accumulates characteristic features of heterochromatin including DNA methylation and inhibitory histone modifications, such as histone H3-lysine 27 trimethylation (H3K27me3) and histone H2A ubiquitination (H2Aub)2. The master regulator of X chromosome silencing is the X-inactivation center (Xic) region, around 100&#8722;500 kb, which controls the counting and pairing of the X chromosomes, the random choice of the X chromosome for inactivation, and the initiation and spreading of silencing along the X chromosome3. The process of X inactivation is initiated by X inactive specific transcript (Xist) that coats the Xi in cis to mediate chromosome-wide silencing and remodel the three-dimensional structure of the X chromosome4. Recently, several proteomic and genetic screens have identified additional regulators of XCI, such as Xist interacting proteins5,6,7,8,9,10,11,12. For example, a previous study using an unbiased genome-wide RNA interference screen identified 13 trans-acting XCI factors (XCIFs)12. Mechanistically, XCIFs regulate Xist expression and therefore, interfering with XCIFs function causes defective XCI12. Together, recent advances in the field have provided important insights into the molecular machinery that is required to initiate and maintain XCI.
Identification of XCI regulators and understanding their mechanism in XCI is directly relevant to X-linked human diseases, such as Rett syndrome (RTT)13,14. RTT is a rare neurodevelopmental disorder caused by a heterozygous mutation in the X-linked methyl-CpG binding protein 2 (MECP2) that affects predominantly girls15. Because MECP2 is located on the X chromosome, RTT girls are heterozygous for MECP2 deficiency with ~50% cells expressing wild-type and ~50% expressing mutant MECP2. Notably, RTT mutant cells harbor a dormant but wild-type copy of Mecp2 on the Xi, providing a source of the functional gene, which if reactivated, could potentially alleviate symptoms of the disease. In addition to RTT, there are several other X-linked human diseases, for which reactivation of Xi represents a potential therapeutic approach, such as DDX3X syndrome.
Inhibition of XCIFs, 3-phosphoinositide dependent protein kinase-1 (PDPK1), and activin A receptor type 1 (ACVR1), either by short hairpin RNA (shRNA) or small molecule inhibitors, reactivates Xi-linked genes12. Pharmacological reactivation of Xi-linked genes is observed in various ex vivo models that include mouse fibroblast cell lines, adult mouse cortical neurons, mouse embryonic fibroblasts, and fibroblast cell lines derived from an RTT patient12. However, whether pharmacological reactivation of Xi-linked genes is feasible in vivo remains to be demonstrated. One limiting factor is the lack of effective animal models to accurately measure the expression of genes from reactivated Xi. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated that carries a genetically labeled Mecp2 on Xi in all the cells due to heterozygous deletion in Xist on the maternal X chromosome16. Using this model, the expression of Mecp2 from Xi has been quantitated following treatment with XCIFs inhibitors in the brain of living mice. Here, the generation of the Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model and methodology to quantitate Xi reactivation in cortical neurons using immunofluorescence-based assays is described.

<table><tbody><tr><td>MICE</td><td></td><td></td><td></td></tr><tr><td>Mecp2&lt;sup&gt;tm3.1Bird&lt;/sup&gt;</td><td>The Jackson Laboratory</td><td>#014610</td><td></td></tr><tr><td>B6;129-Xist (tm5Sado)</td><td>provided by Antonio Bedalov, Fred Hutchinson Cancer Center, Seattle</td><td></td><td></td></tr><tr><td>REAGENTS</td><td></td><td></td><td></td></tr><tr><td>22x22 mm coverslip</td><td>FISHERfinest (Fisher Scientific)</td><td>125488</td><td></td></tr><tr><td>32% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td></td></tr><tr><td>50 ml syringe</td><td>Medline Industries</td><td>NPMJD50LZ</td><td></td></tr><tr><td>60mm culture dish</td><td>CellStar</td><td>628160</td><td></td></tr><tr><td>7-AAD </td><td>BioLegend</td><td>420403</td><td></td></tr><tr><td>ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Fisher 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Labs</td><td>F-1005</td><td></td></tr><tr><td>GSK650394</td><td>ApexBio</td><td>B1051</td><td></td></tr><tr><td>hamilton 10&amp;mu;l syringe </td><td>Hamilton Sigma-Aldrich</td><td>28615-U</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>Ketamine</td><td>Ketaset</td><td>NDC 0856-2013-01</td><td></td></tr><tr><td>Large blunt/blunt curved scissors</td><td>Fine Science Tools</td><td>14519-14</td><td></td></tr><tr><td>LDN193189</td><td>Cayman Chemicals</td><td>11802</td><td></td></tr><tr><td>lodixanol</td><td>Sigma</td><td>1343517</td><td></td></tr><tr><td>magnesium chloride (MgCl2)</td><td>Fisher Chemical</td><td>M35-212</td><td></td></tr><tr><td>Methylcelulose</td><td>Sigma</td><td>M0262-100G</td><td></td></tr><tr><td>mounting medium with DAPI </td><td>Vectashield</td><td>H-1200</td><td></td></tr><tr><td>Needle tip, 26 GA x 1.25&quot;</td><td>PrecisionGlide</td><td>305111</td><td></td></tr><tr><td>ophthalmic ointment </td><td>Refresh Lacri-Lube</td><td>93468</td><td></td></tr><tr><td>optimal cutting temperature (O.C.T.) </td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>PCR mix</td><td></td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin (Pen/Strep)</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Phosphate buffered saline pH 7.4 (PBS)</td><td>Corning Cellgro</td><td>46-103-CM</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>P330-500</td><td></td></tr><tr><td>scalpel blades</td><td></td><td></td><td></td></tr><tr><td>Shallow glass or plastic tray</td><td></td><td></td><td></td></tr><tr><td>skin glue/tissue adhesive</td><td>3M Vetbond</td><td>1469SB</td><td></td></tr><tr><td>sodium azide</td><td>Fisher Scientific</td><td>CAS 26628-22-8</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Chemical</td><td>S642-212</td><td></td></tr><tr><td>standard hemostat forceps</td><td>Fine Science Tools</td><td>13013-14</td><td></td></tr><tr><td>Standard tweezers</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Straight iris scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>sucrose</td><td>Fisher Scientific</td><td>BP220-1</td><td></td></tr><tr><td>Tris-base</td><td>Fisher Bioreagents</td><td>BP152-5</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Bioreagents</td><td>BP151-500</td><td></td></tr><tr><td>Trypsin-EDTA </td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NDC: 59399-111-50</td><td></td></tr><tr><td>EQUIPMENT</td><td></td><td></td><td></td></tr><tr><td>Zeiss AxioObserver Live-Cell microscope </td><td>Zeiss</td><td>Zeiss AxioObserver</td><td></td></tr><tr><td>0.45mm burr</td><td>IDEAL MicroDrill</td><td>67-1000</td><td></td></tr><tr><td>BD FACScalibur</td><td></td><td></td><td></td></tr><tr><td>centrifuge</td><td></td><td></td><td></td></tr><tr><td>glass homogenizer</td><td></td><td></td><td></td></tr><tr><td>cell culture incubator</td><td>Thermo Scientific HERACELL VIOS 160i</td><td>13-998-213</td><td></td></tr><tr><td>Leica 3050S research cryostat </td><td></td><td></td><td></td></tr><tr><td>stereotactic platform </td><td></td><td></td><td></td></tr><tr><td>thermocycler</td><td></td><td></td><td></td></tr><tr><td>Timer</td><td></td><td></td><td></td></tr><tr><td>ultracentrifuge</td><td>Beckman Coulter Optima L-100 XP</td><td></td><td></td></tr><tr><td>Water bath (37 &amp;ordm;C)</td><td>Fisher Scientific Isotemp 2239</td><td></td><td></td></tr></tbody></table>

a non-random mouse model for pharmacological reactivation of mecp2 on the inactive x chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

To demonstrate the feasibility of the Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model for Xi reactivation studies, XCIF inhibitor-mediated reactivation of Xi-linked Mecp2-Gfp was tested in mouse embryonic fibroblasts (MEFs). Female MEFs were isolated from day 15.5 Xist&#916;:Mecp2/Xist:Mecp2-Gfp embryos as described in section 3 (Figure 1A). The genotypes of female Xist&#916;:Mecp2/Xist:Mecp2-Gfp MEFs were confirmed by genotyping-PCR, as de...

Watch this Scientific Journal Video about A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome at JoVE.com

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

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6.3K Views.  University of Virginia School of Medicine. Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

Video: A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Developmental Biology

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Biochemistry

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Medicine

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Behavior

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Immunology and Infection

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Neuroscience

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Summary

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Chapters in this video

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Strategies for Assessing Autistic-Like Behaviors in Mice

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells