Name: modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons at JoVE.com

我们要感谢 "神经发展发展协会" 感谢 jacquier 博士的研究金和 afm-telete通通过 myneuralp 战略计划提供的支持。我们还要感谢克里斯·亨德森博士、威廉·卡穆博士、布里吉特·佩特曼博士、塞德里克·拉乌尔博士和乔治·海斯博士, 他们参与开发和改进了这项技术, 并传播了他们的知识。

所有涉及动物的程序都被该机构的道德委员会接受。
1. 解决方案准备
<ol><li>在 l-15 培养基中制备 4% bsa 透析的10毫升。<ol>
<li>将牛血清白蛋白粉在 l-15 培养基10毫升中, 以 4% (w/v) 的速度溶解。</li>
		<li>将溶液添加到20毫升的透析盒式磁带上, 并提供 20 kda 截止。在4°c 搅拌下, 对 l-15 培养基的500毫升进行透析3天。每天更换 l-15 介质。</li>
		<li>通过0.22μm 膜过滤溶液, 并在 15 ml 管?...

<ol>
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J., 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=Inclusion+body+myopathy+associated+with+Paget+disease+of+bone+and+frontotemporal+dementia+is+caused+by+mutant+valosin-containing+protein.">Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein.</a> Nature Genetics. 36 (4), 377-381 (2004).</li><li>Brown, R. H., Al-Chalabi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyotrophic+Lateral+Sclerosis.">Amyotrophic Lateral Sclerosis.</a> New England Journal of Medicine. 377 (2), 162-172 (2017).</li><li>Taylor, J. P., Brown, R. H., Cleveland, D. 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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=GDNF:+a+potent+survival+factor+for+motoneurons+present+in+peripheral+nerve+and+muscle.">GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.</a> Science. 266 (5187), 1062-1064 (1994).</li><li>Schaller, 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=Novel+combinatorial+screening+identifies+neurotrophic+factors+for+selective+classes+of+motor+neurons.">Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.</a> Proceedings of the National Academy of Sciences. 114 (12), E2486-E2493 (2017).</li><li>Jacquier, 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=Alsin/Rac1+signaling+controls+survival+and+growth+of+spinal+motoneurons.">Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.</a> Annals of Neurology. 60 (1), 105-117 (2006).</li><li>Raoul, C., 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=Chronic+activation+in+presymptomatic+amyotrophic+lateral+sclerosis+(ALS)+mice+of+a+feedback+loop+involving+Fas,+Daxx,+and+FasL.">Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL.</a> Proceedings of the National Academy of Sciences. 103 (15), 6007-6012 (2006).</li><li>Madji Hounoum, B., 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=Wildtype+motoneurons,+ALS-Linked+SOD1+mutation+and+glutamate+profoundly+modify+astrocyte+metabolism+and+lactate+shuttling.">Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.</a> Glia. 65 (4), 592-605 (2017).</li><li>Magrane, J., Sahawneh, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+embryonic+rat+motoneurons+by+panning+on+a+monoclonal+antibody+to+the+low-affinity+NGF+receptor.">Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor.</a> Journal of Neuroscience Methods. 44 (1), 59-70 (1992).</li><li>Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+Isolation+and+Culture+of+Mouse+Embryonic+Motoneurons.">Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li><li>Francius, C., Clotman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+spinal+motor+neuron+diversity:+a+long+quest+for+neuronal+identity.">Generating spinal motor neuron diversity: a long quest for neuronal identity.</a> Cellular and Molecular Life Sciences. 71 (5), 813-829 (2014).</li><li>Neto, 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=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+In+Vitro+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.</a> The Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (1), 16-22 (2004).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Research Notes. 9, (2016).</li><li>Yu, L., 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=Highly+efficient+method+for+gene+delivery+into+mouse+dorsal+root+ganglia+neurons.">Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.</a> Frontiers in Molecular Neuroscience. 8 (2), (2015).</li><li>Malin, S. A., Davis, B. M., Molliver, D. 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该协议的关键点之一是, 在开发过程中, 在精确的时间窗口 (e12.5) 对小鼠胚胎进行解剖, 以优化最终获得的 mn 量。此外, 为了获得最佳产量, 解剖应在上午或下午早些时候进行。在 e12.5 之前 (例如e11.5), 解剖是困难的, 特别是在消除脑膜方面。e12.5 后, 获得的 m2 数量显著下降。为了控制胚胎发育阶段, 成年女性和男性在一天结束时首先被放在一起。第二天早上, 成年人被分开, 女性被检查是否有?...

神经肌肉疾病包括各种临床和基因上不同的疾病, 其特点是肌肉和/或神经系统的改变。由于测序技术的进步, 在过去十年中发现了数百种导致这些罕见疾病的基因 (可在神经肌肉疾病中心查阅的名单, http://neuromuscular.wustl.edu/index.html)。识别突变的多样性表明, 单个基因中的不同突变会导致不同的表型和疾病1、2、3,不同基因的突变会产生相似的表型4 个,5. 在这方面, 正在努力开发能够成为分析突变后果和病理机制的有力工具的细胞模型。
脊髓 mns 有一个大的 somas 位于脊髓的腹侧角, 形成长轴突, 以骨骼肌纤维为目标, 并允许自愿运动, 通过释放乙酰胆碱在神经肌肉交界处。由于 mns 受到肌萎缩侧索硬化症、夏科-玛丽-牙齿疾病 (cmt) 和脊髓肌肉萎缩等神经肌肉疾病的影响, 亨德森博士和他的同事们制定了第一个方案6 , 允许种植体外脊髓 mns 和神经营养因子 gdnf7 (胶质细胞衍生的神经营养因子) 的发现。自那时以来, 技术改进使脊柱 mnm及其亚型能够使用几十年化骨联8进行更准确的纯化, 但密度梯度的浓缩仍然很强大, 并在目前与初级脊柱 mns 合作的实验室中得到广泛使用9,10,11,12,13,14. 随后, 还可以利用表面标记 p75(ntr)15、16、17, 通过免疫调节获得更高的 mn 纯化级。
脊髓包含不同类型的颈椎, 胸椎, 腰椎 m2, 以及中位和侧向运动柱, 不同的位置在前角的背腹轴和目标之间, 他们支配 8,18. 初级 mn 培养可以按生理比例恢复所有这些 mn 亚型。这种技术的主要局限性是在程序结束时获得的 mb 数量少;事实上, 它可以预期从六个胚胎中获得大约10个 5个ms, 这适用于显微镜, 但限制了生物化学实验。为了对更标准化的亚型和丰富的 ms (& gt;106细胞) 进行实验, 胚胎干细胞衍生的 ms 应被考虑到 18。
将野生类型突变基因转染或将内源基因分解为初级 mms 是破译生理病理途径的一种快速而有益的工具, 尤其是在没有小鼠模型的情况下。磁染是一种转染原代神经元的技术, 类似于没有相关神经毒性的脂切。此外, 转染可以在成熟的神经元进行几天后, 在体外, 不同于基于电穿孔9的技术。然而, 这种技术的一个缺点是, 珠子结合核酸的培养, 造成噪音的 dapi 标签。病毒感染可能是最有效的转染 mn 的技术;然而, 磁光化不需要病毒生产和细胞感染所需的某些安全程序。

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone dissection dish</td>
			<td>Living systems instrumentation</td>
			<td>DD-90-S-BLK-3PK</td>
			<td>Sylgard</td>
		</tr>
		<tr>
			<td>round coverslip</td>
			<td>NeuVitro, Knittel glass</td>
			<td>GG-12-Pre</td>
			<td>12 mm</td>
		</tr>
		<tr>
			<td>Slide a Lyzer cassettes</td>
			<td>ThermoFisher Scientific</td>
			<td>66030</td>
			<td>20,000 MWCO ; 30 mL</td>
		</tr>
		<tr>
			<td>Filter unit</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>GP Sterile Syringe Filters</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>4 well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>167063</td>
			<td>Nunclon Delta treated plate</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST by Dumont</td>
			<td>11252-20</td>
			<td>#5 forceps</td>
		</tr>
		<tr>
			<td>scissor</td>
			<td>FST by Dumont</td>
			<td>14060-10</td>
			<td>fine scissors</td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>FST by Dumont</td>
			<td>10035-20</td>
			<td>curved blade</td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>FST by Dumont</td>
			<td>10316-14</td>
			<td>micro-knife scalpel</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>663102</td>
			<td>&#248; x h = 100 x 15 mm</td>
		</tr>
		<tr>
			<td>15 mL polypropylene tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>Watman</td>
			<td>1001125</td>
			<td>circle, 125 mm diameter</td>
		</tr>
		<tr>
			<td>glass chamber slide</td>
			<td>Lab-Tek</td>
			<td>154526</td>
			<td>4 chambers</td>
		</tr>
		<tr>
			<td>Plasmid pCAGEN</td>
			<td>Addgene</td>
			<td>#11160</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions and mediums</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11415056</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 medium, no red phenol</td>
			<td>ThermoFisher Scientific</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Conalbumin</td>
			<td>Sigma-Aldrich</td>
			<td>C7786</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-DL-Ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P8638</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin 2.5%, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca Mg</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td>without Mg2+ Ca2+</td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuron cell culture medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A3582901</td>
			<td>Neurobasal Plus medium</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma-Aldrich</td>
			<td>H6648-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer 1 M</td>
			<td>ThermoFisher Scientific</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradiant medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>Optiprep</td>
		</tr>
		<tr>
			<td>supplement medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A3582801</td>
			<td>B-27 Plus</td>
		</tr>
		<tr>
			<td>Horse serum heat inactivated</td>
			<td>ThermoFisher Scientific</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM</td>
			<td>ThermoFisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>ThermoFisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>penicilline/streptomycine</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immuno fluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>X0515</td>
			<td>without Mg2+ Ca2+</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Acetyl Transferase (CHAT)</td>
			<td>Chemicon</td>
			<td>Ab144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofilament H non phosphorylated (SMI32)</td>
			<td>Biolegends</td>
			<td>SMI-32P</td>
			<td>IF at 1/1000</td>
		</tr>
		<tr>
			<td>Islet-1</td>
			<td>DSHB</td>
			<td>40.2D6</td>
			<td></td>
		</tr>
		<tr>
			<td>Islet-2</td>
			<td>DSHB</td>
			<td>39.4D5</td>
			<td></td>
		</tr>
		<tr>
			<td>Hb9</td>
			<td>DSHB</td>
			<td>81.5C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Lab</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta3 tubulin (Tuj1 clone)</td>
			<td>Biolegends</td>
			<td>801201</td>
			<td>IF at 1/1000</td>
		</tr>
		<tr>
			<td>Lc3b</td>
			<td>Cell Signaling Technology</td>
			<td>#2775</td>
			<td>IF at 1/200</td>
		</tr>
	</tbody>
</table>

modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons

脊髓运动神经元 (mns) 的神经退行性变与大量的神经疾病有关, 包括肌萎缩侧索硬化症、夏科-玛丽-牙齿疾病和脊髓肌肉萎缩, 这些疾病都与肌肉萎缩有关。脊髓 mns 的初级培养已被广泛用于证明特定基因在这类疾病中的参与, 并描述其突变的细胞后果。该协议模拟了一种源自亨德森和他的同事们20多年前的开创性工作的主要 mn 文化。首先, 我们详细介绍了一种从小鼠胚胎中解剖脊髓前角并使用密度梯度从邻近细胞中分离 ms 的方法。然后, 我们提出了一种利用磁光法高效地转换具有表达质粒的 mns 的新方法。最后, 我们说明了如何固定和免疫控制原发性 mns. 利用导致夏科-玛丽-牙齿疾病2型的神经纤维突变, 该协议演示了一种定性的方法来表达感兴趣的蛋白质, 并研究它们的参与mn 的生长、维护和生存。

在培养24小时后, 运动神经元 (mn) 应该已经显示出显著的轴突生长 (至少比 soma 大小长 6倍)。在接下来的几天里, 轴突应该继续生长并显示分支 (图 2)。由于子类型的特殊性, 会有不同的形态。例如, 神经性轴向肌肉的中位柱 mn 比侧向运动柱 mn 具有更短和更多的分支轴突, 而侧向运动柱 mn 则为支配肢体肌肉18。
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Watch this Scientific Journal Video about Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons at JoVE.com

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

这项技术的目的是准备一种高度丰富的来自小鼠脊髓的原发运动神经元 (mns) 培养。为了评估引起 mn 疾病的突变的后果, 我们在这里描述了这些孤立的 m2 的隔离和它们通过磁光的转染。

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

此方法有助于解决神经生物学中的关键问题，如运动神经元极性、轴向、轴翁贩运和神经退行机制。这种技术的主要优点是获得高度丰富的运动神经元培养，同时我们采取表达我们的击倒蛋白质分钟的感情。要开始这个程序，把一个胚胎放在用硅编码的解剖培养皿中。在显微镜下，用钳子将胚胎用腹部对着硅。使用第二对钳子，将其中一个尖端插入罗斯特拉德脊髓的中央运河中。然后，关闭钳子以稍微撕裂后体组织。对康奈尔侧重复此操作，直到打开整个脊髓。接下来，定位胚胎，使开放的脊髓对硅。随后，将脊髓的 rostrad 部分从身体中分离出来。捏住脊髓的 rostrad 部分，然后用头部拉胚胎提取脊髓。小心地拉开仍然附着在脊髓上的任何剩余的脑膜和 DRG。扁平的脊髓在硅上，并使用手术刀，通过沿着每一侧的中间切割去除脊髓的后半。将中间部分切成十块，然后转移到一个新的管子上。要准备脊髓细胞悬浮液，请收集管底的脊髓碎片。用一毫升的火腿 F-10 介质代替 HBSS，并添加 10 微升的三辛。在37摄氏度下孵育管子10分钟。为了停止酶消化，将片段转移到一个新的15毫升管，包含800微升的完整L-15介质，100微升4%BSA，和100微升的DNA和滴定，两次。让碎片沉淀两分钟，然后收集在新鲜15毫升管的上清液。轻轻在上清液管底部加入两毫升 4%BSA。中心在 470 x 重力下熔断 5 分钟。5 分钟后，取出上流液，用 2 毫升完整的 L-15 介质重新悬挂细胞托盘。对于莫托尼龙富集，将细胞悬浮液分成两个15毫升管。然后，向每个管中加入两毫升L-15 PH介质。如果从六个胚胎开始，使用相当于三个胚胎每个管，和三毫升的中等。慢慢添加2毫升的密度梯度溶液，到每个管的底部获得一个尖锐的界面。接下来，在室温下，在 830 x 重力下将中导熔断 15 分钟。在此步骤之后，小细胞应位于管的底部，而大细胞（如莫托龙）应位于密度梯度溶液/介质界面。在接口的细胞中，Asperae 1 至 2 毫升，并将它们转移到新鲜的 15 毫升管中。使用 L-15 PH 介质将音量调整为 10 毫升。随后，在两管底部加入两毫升的4%BSA缓冲。在室温下，在 470 x 重力下进行 5 分钟的中心保险丝。5 分钟后，拆下上经剂，重新悬挂托盘和 3 毫升完整的 L-15 介质。在室温下以 470 x 重力重复离心器 5 分钟。然后，取出上流液，重新悬挂细胞托盘和两毫升的完整培养介质。培养运动神经元，在24个井板中每井500微升培养基中稀释至5000-10000个细胞。然后，在 P-1，000 移液器尖端拆下层溶液。立即转移在培养介质中稀释的运动神经元。编码板，以避免干燥。为了准备DNA，在50微升神经元培养培养的细胞中重新悬浮1微克的DNA。和漩涡5秒。要准备B管，在50微升神经元培养基中重新悬浮1.5微升珠。在50微升DNA溶液中加入50微升珠溶液，在室温下孵育20分钟。在孵育过程中，从将转染的井中提取100微升的培养介质。随后，将100微升的DNA珠混合物转移到每一个井。并在37摄氏度的磁板上孵育24个井板，20-30分钟。这里展示的是现代神经元形态，在培养四天后，内源性非磷酸化神经丝重链的站立所揭示出来。在这些图像中，显示体外四天后的调制解调器神经元形态，并在第二天通过磁体分泌转染为EGFP转基因。座右铭神经元被标记SMI-32或泛神经标记TUJ-1反染色，箭头指示神经元的母细胞。这些是磁感染运动神经元的共合图像，表达野生类型或突变EGFP NEFH构造，并染色为他们的自噬标记LC-3B。箭头显示突变EGFP NEFH蛋白聚合体，也是LC-3B阳性后，蒙托安达库查可以通过视频显微镜分析，以可视化轴子贩运，并添加一些指导。在最初开发解决神经生物学的基本问题后，该技术也被证明是一个强大的工具，探索生理病理机制，涉及莫托龙疾病的光谱，如毒性休克综合征疾病，萨缪尔萎缩性侧索硬化症，或脊柱肌肉萎缩。

Research

JoVE Journal

Neuroscience

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

在癌神经侵袭的体外模型：背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

小鼠模型中风：光血栓形成的焦皮质病变

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...