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 Antoni...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+histological+staining+and+fluorescence+using+ImageJ.">Quantitative analysis of histological staining and fluorescence using ImageJ.</a> Anatomical Record (Hoboken). 296 (3), 378-381 (2013).</li><li>Cseke, L. J., Talley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR-based+genotyping+method+to+distinguish+between+wild-type+and+ornamental+varieties+of+Imperata+cylindrica.">A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.</a> Journal of Visualized Experiments. (60), (2012).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >MICE</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Mecp2tm3.1Bird</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 (NH4Cl)</td>
			<td >Fisher Chemical</td>
			<td >A661-3</td>
			<td></td>
		</tr>
		<tr >
			<td >anti-GFP-AlexaFluor647</td>
			<td >Invitrogen</td>
			<td >A-31852</td>
			<td></td>
		</tr>
		<tr >
			<td >anti-MAP2</td>
			<td >Aves Labs</td>
			<td >MAP</td>
			<td></td>
		</tr>
		<tr >
			<td >BSA</td>
			<td >Promega</td>
			<td >R396D</td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine SR</td>
			<td >Zoopharm</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >citric acid </td>
			<td >Sigma</td>
			<td >C-1857</td>
			<td></td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >Fisher Bioreagents</td>
			<td >BP231-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td >Corning Cellgro</td>
			<td >10-013-CV</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >Decon Labs</td>
			<td >2701</td>
			<td></td>
		</tr>
		<tr >
			<td >fetal bovine serum (FBS)</td>
			<td >VWR Life Science</td>
			<td>89510-198</td>
			<td></td>
		</tr>
		<tr >
			<td >gelatin</td>
			<td >Sigma-Aldrich</td>
			<td >G9391</td>
			<td></td>
		</tr>
		<tr >
			<td >glass slides</td>
			<td >Fisherbrand</td>
			<td >22-034-486</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken FITC-labeled secondary antibody </td>
			<td >Aves 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&#956;l syringe </td>
			<td >Hamilton Sigma-Aldrich</td>
			<td >28615-U</td>
			<td></td>
		</tr>
		<tr >
			<td >Hank&#39;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&#34;</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 &#186;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...

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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 ...

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.

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6.2K Views.  University of Virginia School of Medicine. Questo protocollo può essere utilizzato per generare un modello di topo non casuale per testare e quantificare la riattivazione del cromosoma Mecp2 inattivo legato all'X nel cervello di un topo vivente. Questo modello può essere facilmente modificato per studiare la riattivazione di Xi in altri modelli di malattia legati all'X come la sindrome DDX3X. Sono un post doc nel laboratorio di Sanchita Bhatnagar e presenterò la procedura con Zeming Zheng.Inizia posizionando il mouse anestet...