Name: analysis of translation in the developing mouse brain using polysome profiling
Uploaded: 2021-05-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Translation in the Developing Mouse Brain using Polysome Profiling at JoVE.com

这项工作由国家SERC发现基金（RGPIN/04246-2018至纽约）资助。G.Y.是加拿大研究主席。S.K. 由米塔克斯全球研究生奖学金和 ACHRI 研究生奖学金资助。

所有动物的使用都由卡尔加里大学动物护理委员会监督。用于实验的CD1小鼠是从商业供应商那里购买的。
1. 准备解决方案
注意防止RNA降解，喷洒工作台和所有设备与RNASE净化溶液。实验中使用无鼻塞提示。所有解决方案均在无 RNase 水中准备。
<ol><li>在 DMSO 中准备环氧化物库存解决方案 （100 毫克/mL），并存储在 -20 °C。</li>
	<li>通过在无 RNase 水中加入 75...

<ol>
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多体特征分析是一种常用和强大的技术，用于评估单个基因和全基因组14 级的转化状态。在本报告中，我们提出了一个多体分析协议，使用家庭组装的平台及其应用来分析正在发育的鼠标皮层。这个经济高效的平台易于组装和生成坚固、可重复的蔗糖梯度和具有高灵敏度的多体剖析。
值得注意的是，制备一致和优质蔗糖梯度对于获得可重复的多体剖析结果<s...

在哺乳动物大脑发育过程中，神经干细胞增殖和分化产生神经元和胶质1，2。这个过程的扰动会导致大脑结构和功能的改变，如许多神经发育障碍3，4。神经干细胞的正确行为需要特定基因5的精心表达。虽然对这些基因的表观遗传学和转录控制进行了深入的研究，但最近的发现表明，其他层面的基因调控也有助于协调神经干细胞增殖和分化6、7、8、9、10。因此，解决转化控制程序将极大地促进我们对神经干细胞命运决定和大脑发育背后的机制的理解。
三种具有不同优势的主要技术已被广泛应用于评估 mRNA 的转化状态，包括核糖体分析、翻译核糖体亲和力纯化 （TRAP） 和多体分析。Ribosome 分析使用 RNA 测序来确定受核糖体保护的 mRNA 片段，从而允许对每个成绩单上转译核糖体的数量和位置进行全球分析，通过将其与成绩单丰度11进行比较间接推断翻译率。TRAP利用表位标记的核糖体蛋白来捕获与核糖体结合的mRNA12。鉴于标记的核糖核蛋白可以使用遗传方法在特定的细胞类型中表达，TRAP 允许以特定于细胞类型的方式分析翻译。相比之下，多体分析，它使用蔗糖密度梯度分馏分离自由和翻译不良的部分（较轻的单体）与那些被积极翻译的核糖体（较重的多体），提供了直接测量核糖体密度在mRNA13。这项技术提供的一个优点是其多功能性，以研究翻译特定的mRNA的兴趣，以及全基因组的转学分析14。
本文描述了一个详细的多体特征分析方案，以分析正在发育的老鼠大脑皮层。我们使用家庭组装系统来准备蔗糖密度梯度，并为下游应用收集分数。这里提出的协议可以很容易地适应分析其他类型的组织和生物体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL RNA free microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Greiner-Bio</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>Invitrogen</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>21-23G needle</td>
			<td>BD</td>
			<td>305193</td>
			<td></td>
		</tr>
		<tr>
			<td>2M KCl</td>
			<td>Invitrogen</td>
			<td>AM8640G</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL syringe</td>
			<td>BD</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end needle</td>
			<td>VWR</td>
			<td>20068-781</td>
			<td></td>
		</tr>
		<tr>
			<td>Breadboard</td>
			<td>Thorlabs</td>
			<td>MB2530/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>115-39-9</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 mouse</td>
			<td></td>
			<td>Charles River Laboratory</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved tip forceps</td>
			<td>Sigma</td>
			<td>#Z168785</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td></td>
			<td>Sigma</td>
			<td>66-81-9</td>
		</tr>
		<tr>
			<td>Data acquisition software TracerDAQ</td>
			<td>Measurement Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital converter</td>
			<td></td>
			<td>Measurement Computing</td>
			<td>USB-1208LS</td>
		</tr>
		<tr>
			<td>Direct-zol RNA miniprep kit</td>
			<td>Zymo</td>
			<td>R2070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Bio-basic</td>
			<td>12-03-3483</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Bioshop</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No.5 forceps</td>
			<td>Sigma</td>
			<td>#F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Bio-Rad</td>
			<td>Model 2110</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Wisent</td>
			<td>311-513-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear stage actuator</td>
			<td>Rattmmotor</td>
			<td>CBX1605-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxima first strand cDNA synthesis kit</td>
			<td>Themo Fisher</td>
			<td>M1681</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature V-clamp</td>
			<td>Thorlabs</td>
			<td>VH1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series breadboard</td>
			<td>Thorlabs</td>
			<td>MSB7515/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series optical post</td>
			<td>Thorlabs</td>
			<td>MS2R/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series pedestal post holder base</td>
			<td>Thorlabs</td>
			<td>MBA1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio-basic</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#216;12.7 mm aluminum post</td>
			<td>Thorlabs</td>
			<td>TRA150/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR green fastmix</td>
			<td>Quanta Bio</td>
			<td>CA101414-274</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Bioshop</td>
			<td>58-58-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Right-angle clamp</td>
			<td>Thorlabs</td>
			<td>RA90/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Right-angle &#216;1/2&#34; to &#216;6 mm post clamp</td>
			<td>Thorlabs</td>
			<td>RA90TR/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase AWAY</td>
			<td>Molecular BioProducts</td>
			<td>7002</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase free tips</td>
			<td>Frogga Bio</td>
			<td>FT10, FT200, FT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>Wisent</td>
			<td>809-115-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>Slim right-angle bracket</td>
			<td>Thorlabs</td>
			<td>AB90B/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Small V-clamp</td>
			<td>Thorlabs</td>
			<td>VC1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>302-95-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stepper motor driver</td>
			<td>SongHe</td>
			<td>TB6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Bioshop</td>
			<td>57501</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Bio-basic</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermofisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube piercer</td>
			<td>Brandel</td>
			<td>BR-184</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td></td>
			<td>Beckman Coulter</td>
			<td>L8-70M</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1M Tris-HCl pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>UNO project super starter kit</td>
			<td>Elegoo</td>
			<td>EL-KIT-003</td>
			<td></td>
		</tr>
		<tr>
			<td>UV monitor</td>
			<td>Bio-Rad</td>
			<td>EM-1 Econo</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical bracket</td>
			<td>Thorlabs</td>
			<td>VB01A/M</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation in the developing mouse brain using polysome profiling

哺乳动物大脑的正常发育依赖于神经干细胞增殖和分化成不同神经细胞类型的精细平衡。这种平衡由基因表达紧密控制，基因表达在多个级别进行微调，包括转录、转录后和翻译。在这方面，越来越多的证据突出了转化调节在协调神经干细胞命运决定方面的关键作用。多体分馏是评估全球和单个基因水平的 mRNA 转化状态的有力工具。在这里，我们提出了一个内部聚体分析管道，以评估细胞的转化效率从正在发育的老鼠大脑皮层。我们描述了蔗糖梯度制备、组织裂解、超中心化和基于分数的 mRNA 转化状态分析的协议。

作为证明，含有75μgRNA（从8个胚胎中汇集）的皮质分析被蔗糖梯度分成12个分数。紫外线吸收峰值在254纳米识别分数包含40S亚单位，60S亚单位，80S单体和多体（图4A）。Rpl10通过对大型核糖体亚单位的西色斑点进行分析，显示其存在60S子单元（分数3）、单体（分数4）和多体（分数5-12）（图4B）。相比之下，细胞质蛋白Gapdh和Csde1与核糖体无关，而是富含?...

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Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

哺乳动物大脑的发展需要在翻译水平上正确控制基因表达。在这里，我们描述了一个多体分析系统，该系统具有易于组装的蔗糖梯度制作和分馏平台，以评估 mRNA 在发育中的大脑中的转化状态。

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

转化调节在大脑发育中起着重要的作用。多体分析提供了一个有用和强大的工具来评估mRNA的转化状态，使得在大脑发育过程中检查转化控制功能成为可能。这种方法是一种简单和低成本的方法，使蔗糖梯度和评估多体分馏。在此协议中，它用于分析正在发育的鼠标皮层。然而，它可以很容易地适应研究其他系统。首先稀释2.2摩尔蔗糖，准备6个10至50%蔗糖梯度。用大约 16 毫升的 10% 蔗糖填充 30 毫升注射器，将其放在注射器泵上并将其连接到针头。确保注射器、管子或针头中没有气泡，并擦拭针尖以去除任何残留溶液。将电动舞台向上移动，使针尖接触底部管的中心。将注射器泵设置为每分钟两毫升的流量，体积为 2.3 毫升。分配 2.3 毫升 10% 蔗糖溶液后，向下移动机动阶段，并重复所有六个梯度的过程。然后在管子底部加入20%蔗糖溶液，然后在第12天收集30、40和50%收集CD1小鼠胚胎，并将它们放在含有冰冷HBSS的10厘米板中。在解剖范围下，将一个胚胎转移到一个含有冰冷HBSS的6厘米板上。使用 21 到 23 个仪表针，通过 45 度角穿透眼睛来固定头部位置，并施加力确保针头固定在盘子上。使用五号钳子去除从中间到两侧工作的皮肤和头骨，然后切开嗅球，去除脑膜，露出皮质组织。使用弯曲的钳子将皮质组织切成冰上两到三毫升的神经巴萨介质。根据需要从不同的胚胎中提取组织。从8个胚胎到10个胚胎的组织通常给出大约200微克的总RNA。将环丙酰胺加入神经巴氏介质，将解剖组织最终浓度为每毫升100微克，并在37摄氏度下孵育10分钟。在 500 x g 下将组织在摄氏 4 度下分离 5 分钟，然后丢弃超自然人。用冰冷的PBS两次清洗组织，并辅以每毫升环丙酰胺100微克。加入500微升细胞裂解缓冲器，并辅以四微升的RNase抑制剂和移液器上下，以恢复组织。使用胰岛素针轻轻解洗组织。然后在冰上孵育10分钟，每两到三分钟短暂旋转一次。孵化后，在2000×g下将离心解水在摄氏4度下5分钟，并将超自然物转移到冰上新的离心管。在 13，000 x g 时再次重复此过程离心。将超自然物转移到冰上的新离心管后，使用紫外线-维斯光谱仪测量组织测定液中的RNA浓度。将利萨酸盐慢慢分配到超中轴管的墙壁上，将样品加载到蔗糖梯度上。轻轻地将蔗糖梯度放在桶中，并在 19 万 x g 和 4 摄氏度下将样品置于超中心，持续 90 分钟。在管穿孔器上放置一个空的超中心管，然后用针头从底部轻轻穿透管子。用 25 毫升 60% 蔗糖追逐溶液填充 30 毫升注射器。然后轻轻按压注射器，使追逐液填充空管，并通过 254 纳米紫外线监视器设置基线进行检测。在紫外线监视器上按自动零度以注册基线进行检测，然后按"播放"软件开始录制。一旦系统建立基线，暂停在软件上录制并收回追逐解决方案，确保系统中没有残余的追逐解决方案。将一个样品管加载到管穿孔器上，然后用针头从底部轻轻穿透管子。开始在软件上录制，确保线路中没有气泡。然后启动注射器泵和分数收集器收集多体分数，将分水器设置为 30 秒。将分数收集到 1.5 毫升管中。从8个胚胎中提取的含有RNA的皮质分析液通过蔗糖梯度分离成12个分数。254纳米的紫外线吸收峰值确定了包含40S亚单位60S亚单位和80S单体和多体的分数。对大型核糖体亚单位 Rpl10 的西方污点的分数的分析表明，它存在 60S 亚单位单体和聚体。相比之下，细胞质蛋白Gapdh和Csde1与核糖体无关，而是富含含有自由RNA的分数。与不同分数的蛋白质分离一致，Gapdh 和 sox2 mRNA 在含有重聚体的分数中高度丰富，表明这些 mRNA 在发育中的皮层中得到了有效的转化。相比之下，rpl7和rpl35 mRNA在包含单体的分数中得到了丰富，这表明了压抑的翻译。为了获得可重复的结果，在制作蔗糖梯度时保持一致性非常重要。注射器泵应用于喷射蔗糖。在分馏和样品收集过程中，使用无 RNase 水清洗系统非常重要。这减少了前一个样本中剩余的蔗糖。此方法可用于评估各种组织中 mRNA 的转化状态。利用该平台，可以获得聚体分析的一致梯度，这为这种经典而有用的技术提供了低成本的解决方案。

Research

JoVE Journal

Neuroscience

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Single-cell Profiling of Developing and Mature Retinal Neurons

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...