Name: a non-random mouse model for pharmacological reactivation of mecp2 on the inactive x chromosome
Uploaded: 2019-05-22T14:00:00
Description: Watch this Scientific Journal Video about A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome at JoVE.com

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...

<ol>
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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 ...

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