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

提交人感谢 Antonio Bedalov 提供试剂;弗吉尼亚大学冷冻切片组织组织组织组织组织核心;弗吉尼亚大学流式细胞术核心, 用于流式细胞术分析;Christian Blue 和 Saloni Singh 提供基因分型方面的技术援助。这项工作得到了向 z. z. 提供的双 Hoo 研究赠款、维吉尼亚大学弗吉尼亚技术种子基金奖和哈特威尔基金会个人生物医学研究奖的试点项目奖的支持。

涉及老鼠的工作得到了弗吉尼亚大学动物护理和使用机构委员会 (IACUC; #4112) 的批准。
1. 在 Xi 上生成具有基因标记Mecp2的非随机 Xci 鼠标模型
请注意:研究中使用的小鼠株如下: mcmp-2 tm3.1 bird 和xist/ xist (B6;129-Xist<tm5Sado> 由 antonio bedalov、 fred 提供)西雅图哈钦森癌症中心)。在各自菌株之间的育?...

<ol>
	<li>Lyon, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome+inactivation+as+a+system+of+gene+dosage+compensation+to+regulate+gene+expression.">X-chromosome inactivation as a system of gene dosage compensation to regulate gene expression.</a> Progress in Nucleic Acid Research and Molecular Biology. 36, 119-130 (1989).</li><li>Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delving+into+the+diversity+of+facultative+heterochromatin:+the+epigenetics+of+the+inactive+X+chromosome.">Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome.</a> Current Opinion in Genetics Development. 15 (5), 482-489 (2005).</li><li>Augui, S., Nora, E. P., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+X-chromosome+inactivation+by+the+X-inactivation+centre.">Regulation of X-chromosome inactivation by the X-inactivation centre.</a> Nature Review Genetics. 12 (6), 429-442 (2011).</li><li>Pontier, D. B., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xist+regulation+and+function+explored.">Xist regulation and function explored.</a> Human Genetics. 130 (2), 223-236 (2011).</li><li>Barnes, C., Kanhere, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+RNA-Protein+Interactions+Through+In+Vitro+RNA+Pull-Down+Assays.">Identification of RNA-Protein Interactions Through In Vitro RNA Pull-Down Assays.</a> Methods in Molecular Biology. 1480, 99-113 (2016).</li><li>McHugh, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li><li>Minajigi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomes.+A+comprehensive+Xist+interactome+reveals+cohesin+repulsion+and+an+RNA-directed+chromosome+conformation.">Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.</a> Science. 349 (6245), (2015).</li><li>Mira-Bontenbal, H., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Xist-Interacting+Proteins+in+X-Chromosome+Inactivation.">New Xist-Interacting Proteins in X-Chromosome Inactivation.</a> Current Biology. 26 (8), R338-R342 (2016).</li><li>Mira-Bontenbal, H., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Xist-Interacting+Proteins+in+X-Chromosome+Inactivation.">New Xist-Interacting Proteins in X-Chromosome Inactivation.</a> Curren Biology. 26 (10), 1383 (2016).</li><li>Ridings-Figueroa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+matrix+protein+CIZ1+facilitates+localization+of+Xist+RNA+to+the+inactive+X-chromosome+territory.">The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory.</a> Genes and Development. 31 (9), 876-888 (2017).</li><li>Sunwoo, H., Colognori, D., Froberg, J. E., Jeon, Y., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeat+E+anchors+Xist+RNA+to+the+inactive+X+chromosomal+compartment+through+CDKN1A-interacting+protein+(CIZ1).">Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1).</a> Proceedings of National Academy of Sciences of the United States of America. , (2017).</li><li>Bhatnagar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+pharmacological+reactivation+of+the+mammalian+inactive+X+chromosome.">Genetic and pharmacological reactivation of the mammalian inactive X chromosome.</a> Proceedings of National Academy of Sciences of the United States of America. 111 (35), 12591-12598 (2014).</li><li>Zoghbi, H. Y., Percy, A. K., Schultz, R. J., Fill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+X+chromosome+inactivation+in+the+Rett+syndrome.">Patterns of X chromosome inactivation in the Rett syndrome.</a> Brain Development. 12 (1), 131-135 (1990).</li><li>Anvret, M., Wahlstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome:+random+X+chromosome+inactivation.">Rett syndrome: random X chromosome inactivation.</a> Clinical Genetics. 45 (5), 274-275 (1994).</li><li>Amir, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome+is+caused+by+mutations+in+X-linked+MECP2,+encoding+methyl-CpG-binding+protein+2.">Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.</a> Nature Genetics. 23 (2), 185-188 (1999).</li><li>Przanowski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+reactivation+of+inactive+X-linked+Mecp2+in+cerebral+cortical+neurons+of+living+mice.">Pharmacological reactivation of inactive X-linked Mecp2 in cerebral cortical neurons of living mice.</a> Proceedings of Natlional Academy of Sciences of the United States of America. 115 (31), 7991-7996 (2018).</li><li>Borensztein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xist-dependent+imprinted+X+inactivation+and+the+early+developmental+consequences+of+its+failure.">Xist-dependent imprinted X inactivation and the early developmental consequences of its failure.</a> Nature Structural and Molecular Biology. 24 (3), 226-233 (2017).</li><li>Jensen, E. 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>

此前, 有选择地需要的 Xcif 对哺乳动物女性细胞中的氧连锁基因进行沉默, 结果被确定为12。我们进一步优化了针对 Xcif 的有效小分子抑制剂, 如 ACVR1 和 PDPK1 的下游效应, 它能有效地激活小鼠成纤维细胞细胞系、小鼠皮质神经元和人成纤维细胞中的氧连锁Mep2从 RTT 患者派生的行。这些结果表明, 习近平的再激活是一种可行的治疗方法, 以挽救 x 连锁疾病患者的基因缺陷;然而, ...

X 染色体失活 (XCI) 是一个剂量补偿的过程, 通过沉默 x 染色体的一个副本 x 染色体在女性1平衡 x 连锁基因表达。因此, 非活性 X 染色体 (Xi) 积累了异染色质的特征特征, 包括 DNA 甲基化和抑制组蛋白修饰, 如组蛋白 H3-lysine 27 三甲基化 (H3K27me3) 和组蛋白 H2A 泛性 (H2Aub)2. x 染色体沉默的主调节器是 x 失活中心 (xic) 区域, 约 100-500kb, 控制 x 染色体的计数和配对, 随机选择 x 染色体的失活, 以及启动和启动和沿 X 染色体3号的沉默传播。X 失活的过程是由 X 不活跃的特异性转录 (xist) 启动的, 它覆盖了西在cis中, 介导全染色体的沉默, 并重新建模 x 染色体4的三维结构。最近, 一些蛋白质组和基因屏幕已经确定了 xci 的其他调节剂,如xist相互作用的蛋白质5,6,7, 8,9 ,10,11,12. 例如, 以前一项使用无偏见的全基因组 rna 干扰屏幕的研究确定了 13个反作用Xci 因素 (xcif)12。在机械上, Xcif 调节Xist 表达,因此, 干扰 xcif 功能会导致有缺陷的 xci12。该领域最近的进展共同为启动和维护 XCI 所需的分子机制提供了重要的见解。
Xci 调节剂的识别和对其在 xci 中的机制的了解与 x 连锁人类疾病直接相关, 例如 rett 综合征 (rtt)13、14。RTT 是一种罕见的神经发育障碍, 由 x 连锁甲基 cpg 结合蛋白 2 (mecp2) 中的杂合突变引起, 主要影响15岁的女孩.由于Mecp2位于 x 染色体上, RTT 女孩对Mecp2缺乏症具有杂合, 其中约50% 的细胞表达野生类型, ~ 50% 表达突变mecp2。值得注意的是, RTT 突变细胞在习近平上有一个休眠但野性的Mcp2副本, 提供了功能基因的来源, 如果重新激活, 可能会缓解这种疾病的症状。除了 RTT, 还有其他几种 x 连锁人类疾病, 其中习近平的再激活代表了一种潜在的治疗方法, 如 DDX3X 综合征。
通过短发夹 Rna (SHRNA) 或小分子抑制剂对 Xcif、3-磷酸苷依赖蛋白激酶-1 (PDPK1) 和激活蛋白 A 受体类型 1 (ACVR1) 的抑制作用, 使氧连接基因12重新激活。在各种体外模型中观察到了与氧相关基因的药理再激活, 这些模型包括小鼠成纤维细胞系、成年小鼠皮质神经元、小鼠胚胎成纤维细胞和来自 RTT 患者12的成纤维细胞系。然而, 与西有关的基因在体内的药理再激活是否可行还有待证明。一个限制因素是缺乏有效的动物模型来准确测量反应新激活后的基因表达。为了实现这个目标, 一个Xisti:mcmp2-g夫 p小鼠模型已经生成, 由于在母亲 x 染色体16上的 xist 中的杂合缺失, 在所有细胞中携带一个基因标记的 mcp2 在 xi 上. 利用该模型, 在活鼠大脑中使用Xcif 抑制剂治疗后, 对习近平 mecp2 的表达进行了定量处理。在这里, 介绍了基于免疫荧光的检测方法, 对皮质神经元中 xi 再激活进行定量的模型和方法。

<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 染色体失活 (XCI) 是女性中一个 X 染色体的随机沉默, 以实现性别之间的基因剂量平衡。因此, 所有雌性都是杂合体的 x 连锁基因表达。XCI 的关键调节器之一是xist, 这对于 xci 的启动和维护至关重要。先前的研究已经确定了13个反式作用 X 染色体失活因子 (Xcif) 使用一个大规模的, 功能丧失的基因筛选。使用短发夹 RNA 或小分子抑制剂抑制 Xcif, 如 ACVR1 和 PDPK1, 可在培养细胞中重新激活 X 染色体相关基因。但在体内重新激活不活跃 x 染色体的可行性和耐受性仍有待确定。为了实现这个目标,一个 Xisti:mcgho/xits:micp2-g夫p 小鼠模型是用非随机 xci 生成的, 因为在一个 x 染色体上删除了 xist 。利用该模型, 用 XCIF 抑制剂治疗后, 小鼠大脑中的非活性 x 再激活程度被定量。最近公布的结果表明, Xcif 的药理抑制首次从活着的小鼠大脑的皮质神经元的不活跃 x 染色体中重新激活Mep2 。

为了证明Mésti:mcgp2-g夫小鼠模型在 xi 再激活研究中的可行性, 在小鼠胚胎成纤维细胞 (mef) 中测试了 xcif 抑制剂介导的西联Mcp-2-gfp 再激活。如第3节 (图 1a) 所述, 从 15.5天开始分离女性 mef: mcp2·xit:mcp2-g夫胚胎。女性水族的基因型: Micp2/xite:mc常见-gfp 多元醇通过基因型 pcr 证实, 如前面所述的 19 (图 1b) 和基于...

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

在这里, 我们描述了一个协议, 以生成一个可行的女性小鼠模型与非随机 X 染色体失活, 即, 母体遗传 x 染色体是不活跃的100% 的细胞。我们还描述了一个方案, 以测试可行性, 耐受性和安全性的药理作用, 在体内的非活性 X 染色体。

非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

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 ...

该协议可用于生成非随机小鼠模型，用于测试和量化活鼠大脑中不活跃的 X 链接 Mecp2 染色体的重新激活。此模型可以很容易地修改，以研究其他 X 相关疾病模型（如 DDX3X 综合征）中的 Xi 重新激活。我是桑奇塔·巴特纳加尔实验室的博士后，我将和郑泽民一起演示手术过程。首先将麻醉鼠标定位在立体定向平台中，将切口钩在鼻子抑制器的咬杆上。拧紧鼻夹，同时保持鼠标头在水平平面上。调整耳杆的高度以到达耳道的耳道部分，以保持头部固定。然后用局部防腐剂和70%乙醇的交替擦拭对头部进行消毒。当鼠标准备好时，使用无菌手术刀在头皮中间做一个0.75厘米的水平切口。使用 0.45 毫米毛刺在右和左皮质半球上方钻两个对称孔，从下垂缝合线钻两毫米，在左骨的近似中间钻两毫米的羊皮缝合线。将 10 微升注射器牢固地连接到立体定向平台，将 10 微升新准备的化学抑制剂加载到注射器中。将针头推进到第一个毛刺孔中，同时保持针头垂直于头骨。当针穿过头骨时，将立体定向数字显示屏上的坐标归零，并推进针头的尖端，直到其深度达到 2.5 毫米。将针头提取0.5毫米至两毫米的深度，并在一分钟内缓慢注入溶液的整个10微升体积。所有抑制剂都送来后，将针头留在大脑中再留一分钟，然后取走针头。仅使用车辆作为控制装置将注射到第二个毛刺孔中，并关闭切口上的皮肤。然后将鼠标从立体声装置转移到 37 摄氏度的加热垫上，并进行监控，直到完全恢复。在剂量方案结束时，将鼠标固定到解剖垫上。使用剪刀和钳子，通过肋骨下方的切口和腹壁进行横向切口。小心地将肝脏与隔膜分离，沿着肋骨的整个长度切开隔膜，露出胸腔。使用剪刀将切口切到左心室的后端，并在两分钟内立即用大约 15 毫升 PBS 注射右心室。成功的灌注将导致肝脏颜色从红色变成淡粉色。当所有 PBS 已交付后，在两分钟内用 PBS 中约 10 毫升的 4% 甲状甲醛来给心脏。收获头部后，用剪刀在头皮上做中线切口，露出头骨。将剪刀尖放入前脑大体中，以方便在头骨中向眼睛创建横向切口。在头骨的另一侧进行类似的切口，使剪刀的端尽可能肤浅，并切割头骨在眼睛之间和鼠标鼻子上方的区域。使用钳子轻轻地剥下脑半球的颅骨，用铲子抬高大脑。仔细解剖将大脑固定到头骨并放入塑料盘中的颅神经纤维。然后切除小脑和嗅觉球。为了获得脑组织的部分，首先在PBS中充满4%半成甲醛的15毫升管中固定大脑，在4摄氏度过夜。第二天早上，每次洗涤新鲜 PBS，至少冲洗脑组织三次，5分钟，温度为4摄氏度。最后一次洗涤后，用剪刀和钳子拆下每个半球的前部，将样品放入标有低温模具的正面向下。将每个组织淹没在最佳切割温度介质中，然后将样品在 10 毫米塑料 Petri 盘中冷冻，以存放零下 80 摄氏度。要确定Xi的重新活化，将5至6微米的脑组织部分浸入抗原检索溶液中，在100摄氏度的热块上浸泡5分钟。在孵化结束时，在室温下用四个五分钟洗涤，每次洗涤新鲜 PBS，然后将幻灯片浸入阻尼溶液中。在室温下20分钟后，每次洗涤用新鲜洗涤缓冲液洗三次幻灯片，5分钟。在四摄氏度下将组织样本与适当的原抗体在四摄氏度下染色过夜。在荧光显微镜上拍摄幻灯片，调整对比度和亮度，以便量化药物和车辆治疗小鼠脑半球的 GFP 阳性细胞数量。为了证明非随机X染色体灭活小鼠模型对于非活动X染色体再活化研究的可行性，通过基因分型PCR和流细胞切除术确认了雌性转基因小鼠胚胎成纤维细胞的基因型。经PCR评估，GFP标记甲基氯联苯基蛋白的表达两个基因或 MECP2 被药物重新激活，而不是车辆处理。非随机X染色体灭活小鼠模型胚胎成纤维细胞也表示核GP后药物，但没有车辆治疗证明X染色体灭活因子抑制剂重新激活灭活X染色体链接MCP2在小鼠胚胎成纤维细胞。药物治疗在药物注入的大脑半球中大约30%的细胞中重新激活灭活X染色体 MECP2-GFP，而在车辆注入半球中未检测到重新激活。微管相关蛋白两个阳性神经元在药物治疗半球中也出现GP阳性，表明有灭活X染色体MCP2-GFP表达。药物方案将取决于小分子抑制剂的目标和疗效，因此在体内测试之前优化体外剂量和治疗。非随机小鼠模型可用于单独或组合测试不同的药物。事实上，我们和其他人已经发现了几种可药物的X染色体失活因子。

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SCIENTISTS IN ACTION

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

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

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

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

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

使用果蝇的异色素促进药物的鉴定筛选方法

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

通过定量染色体形成捕获（4C）鉴定胚胎体增强剂-促进者接触

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model

利用小鼠模型研究MYH7突变Gly823Glu在家族性肥厚型心肌病中的发病机制

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM ...