Name: modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes
Uploaded: 2019-12-20T12:00:00
Description: Watch this Scientific Journal Video about Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes at JoVE.com

这项工作得到了斯库拉师范高级院（SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIOSNS16_B_DIPRIMIO）。

1. 细胞培养
<ol><li>培养SH-SY5Y细胞（人类神经母细胞瘤细胞系，CRL-2266）在完整的介质（Dulbeco的改性鹰培养基：营养混合物F12[DMEM/F-12]补充10%胎儿牛血清[FBS]，2mM L-谷氨酰胺，100 U/mL青霉素和100微克/mL链霉素）。将孵化器中的细胞保持在37°C和5%CO2。在10厘米的板中生长细胞，并在汇入时分裂。</li>
</ol>2. 细胞分化
<ol><li>为了区分SH-SY5Y细胞，在电镀后的第二天，加入10μM视?...

<ol>
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我们描述了一种测量核Tau蛋白对基因表达影响的方法。有了这个协议，细胞质陶的贡献是强烈的限制。该协议的关键步骤如下：人类神经母细胞瘤SH-SY5Y细胞的分化、亚细胞分馏和Tau蛋白在核腔中的定位。
首先，如代表性结果部分所示，通过添加RA和BDNF来分化SH-SY5Y细胞对于在培养中获得神经元样细胞的良好制备至关重要。播种的细胞密度特别重要，因为较低的密度可能会影响...

Tau蛋白在核中的功能近年来引起了极大的兴趣，因为它已被证明与核酸1，2，3，4，5，6密切相关。事实上，最近的一项全基因组研究表明，Tau在体内结合基因和基因DNA序列。在核组织中的作用已被建议8，9，10，11。此外，Tau被建议参与DNA保护，免受氧化和高热应激5、10、12、13的影响，而突变的陶已与染色体不稳定和抗倍体14、15、16有关。
直至目前为止，研究陶在核舱的功能方面的挑战，由于难以剖析核陶在核质Tau的贡献中的具体贡献，因此，研究陶在核舱中所面临的挑战，几乎仍未解决。此外，迄今为止，核隔间中的Tau分子的功能只是相关的，因为它们缺乏核Tau蛋白直接参与的明确证明。事实上，陶参与逆子的流动或rRNA转录或DNA保护11、12、17、18、19，也可以由细胞质陶的贡献或与核陶无关的其他下游因素的影响来解释。
在这里，我们提供了一个方法，可以通过利用一个经典程序来隔离核隔间，结合使用带有核定位（NLS）或核出口信号（NES）标记的Tau构造0N4R来解决这个问题。这种方法消除了由于Tau分子从细胞质室溢出而可能产生的文物的复杂问题。此外，Tau-NLS和Tau-NES结构分别诱导Tau分子从核隔间中浓缩或排除，为核Tau分子参与特定功能提供正负控制。该协议在技术上是容易的，它由经典和可靠的方法组成，广泛适用于研究Tau在其他细胞类型中的核功能，有区别与否，如重新激活Tau表达20，21的癌细胞。此外，它也可以应用于细胞质和细胞核中存在的其他蛋白质，以解剖与不同隔间相关的生物功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 633 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td>IF 1:500</td>
		</tr>
		<tr>
			<td>anti Actin Antibody</td>
			<td>BETHYL LABORATORIE</td>
			<td>A300-485A</td>
			<td>anti-rabbit WB 1:10000</td>
		</tr>
		<tr>
			<td>anti GAPDH Antibody</td>
			<td>Fitzgerald Industries International</td>
			<td>10R-G109a</td>
			<td>anti-mouse WB 1:10000</td>
		</tr>
		<tr>
			<td>anti H2B Antibody</td>
			<td>Abcam</td>
			<td>ab1790</td>
			<td>anti-rabbit WB 1:15000</td>
		</tr>
		<tr>
			<td>anti Tau-13 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-21796</td>
			<td>anti-mouse WB 1:1000; IF 1:500</td>
		</tr>
		<tr>
			<td>anti Tubulin alpha Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-16891</td>
			<td>anti-mouse WB 1:5000</td>
		</tr>
		<tr>
			<td>anti VGluT1 Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>AMAb91041</td>
			<td>anti-mouse WB 1:500</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Euroclone</td>
			<td>EMPO14500</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Alomone Labs</td>
			<td>B-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting-Grade Blocker</td>
			<td>Biorad</td>
			<td>1706404</td>
			<td>Non-fat dry milk</td>
		</tr>
		<tr>
			<td>BOVIN SERUM ALBUMIN</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>Criterion TGX 4-20% Stain Free, 10 well</td>
			<td>Biorad</td>
			<td>5678093</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>GIBCO</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium Low Glucose</td>
			<td>Euroclone</td>
			<td>ECM0060L</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>0390-100ml</td>
			<td>pH=8 0.5M</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Euroclone</td>
			<td>EC50182L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2004</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-50ml</td>
			<td>Octylphenoxy poly(ethyleneoxy)ethanol</td>
		</tr>
		<tr>
			<td>Immobilon Western</td>
			<td>MERCK</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tech Chamber slide 8 well glass slide</td>
			<td>nunc</td>
			<td>177402</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12566014</td>
			<td>cationic lipid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-6X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma-Aldrich</td>
			<td>40,872-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/ml, 100ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Dulbecco A)</td>
			<td>OXOID</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop</td>
			<td>Roche</td>
			<td>4906837001</td>
			<td>phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Step 3.10</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Subcellular Protein Fractionation Kit for cultured cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>78840</td>
			<td></td>
		</tr>
		<tr>
			<td>Supported Nitrocellulose membrane</td>
			<td>Biorad</td>
			<td>1620097</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-Plate 6well</td>
			<td>SARSTEDT</td>
			<td>833,920</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP2 laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ml</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Plus SV Minipreps DNA Purification Systems</td>
			<td>Promega</td>
			<td>A1330</td>
			<td>Step 3.5</td>
		</tr>
	</tbody>
</table>

modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes

Tau是一种在神经元中表达的微管结合蛋白，其主要已知功能与维持细胞骨骼稳定性有关。然而，最近的证据表明，Tau也存在于其他亚细胞室中，包括其与DNA保护、rRNA转录、逆转录泊剂流动性和结构组织有关的细胞核。核仁。我们最近已经证明，核Tau参与VGluT1基因的表达，建议一种分子机制，可以解释谷氨酸释放在阿尔茨海默氏病的早期阶段的病理增加。直到最近，由于技术限制，防止了细胞质Tau的贡献或其它影响，核陶参与调节目标基因的表达一直相对不确定和模糊。下游因素与核陶无关。为了克服这种不确定性，我们开发了一种方法，研究由核Tau蛋白专门调节的目标基因的表达。我们采用了一种协议，将定位信号和亚细胞分馏的使用耦合在一起，从而排除了细胞质Tau分子的干扰。最值得注意的是，该协议是容易的，由经典和可靠的方法组成，广泛适用于研究Tau在其他细胞类型和细胞条件下的核功能。

图1描述了用于剖析核陶在基因表达中避免细胞质Tau蛋白贡献的策略。简单地说，标有NLS或NES标记的Tau蛋白分别在核隔间中积累或排除。这种不平衡的功能效应是作为VGluT1基因产物测量的基因表达的改变。
按照协议描述，SH-SY5Y细胞用RA处理5天，然后用BDNF治疗3天，以获得后线神经样细胞（?...

Watch this Scientific Journal Video about Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes at JoVE.com

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau 是一种神经元蛋白，存在于细胞质中，它结合微管，在细胞核中，它发挥非常规功能，包括与阿尔茨海默氏病相关基因的调制。在这里，我们描述了一种研究核Tau功能的方法，同时排除了来自细胞质陶的任何干扰。

陶子细胞定位作为研究疾病相关基因表达的工具的调制

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

这种方法可以帮助回答有关tau在核舱中直接作用的关键问题，同时排除任何潜在的细胞质tau污染。该技术的主要优点是，它广泛适用于研究tau在其他细胞类型和不同细胞条件下的核功能。演示程序将是贾科莫西亚诺，一个后医生从我的实验室。首先将四乘十十至五个SH-SY5Y人类神经母细胞细胞系细胞与空对照载体进行转染，四乘十至五细胞转染与未标记的tau，四乘十至五细胞转染为tau-NLS，四乘十至五细胞与tau-NES转染成六孔板的单个孔。电镀后的第二天，分别孵育400南克的DNA和适当的量阳离子脂到一管250微升减少血清每井。在室温下五分钟后，将每个载体与一卷阳离子脂结合，并在室温下孵育DNA脂质复合物20分钟。在孵育结束时，用两毫升新鲜完整培养基代替每井的超级钠，并转染每井，用适当的DNA脂质复合溶液在37摄氏度下进行隔夜孵育。第二天，用新鲜的分化介质代替超级钠。对于西叶分析，用PBS洗涤每井中转染和分化的细胞，并在37摄氏度下用500微升0.1%的三叶草孵育细胞，四分钟。当细胞分离后，停止反应，用等量的完整介质，将细胞悬浮液从每个井转移到单独的管中进行离心。小心去除超钠后，将颗粒重新浸入每管一毫升 PBS 中的颗粒进行第二次离心，然后再次去除超自然剂。对于总蛋白提取物，在50至100微升的解解缓冲液中孵育颗粒30分钟，并辅以蛋白酶和磷酸酶抑制剂。在孵化结束时，将提取物以16，000次g离心15分钟，通过任何标准定量测定来量化蛋白质浓度。然后将每个蛋白质样品的20微克与5微升的4X Laemmli缓冲液混合到20微升的总体积中，并在100摄氏度下煮沸5分钟。对于亚细胞分馏，重新使用完整介质重新暂停细胞以进行计数，并向下旋转 10 倍到每个样本的 6 个细胞。为了分离细胞酸分，孵育100微升冰冷细胞质提取缓冲液中的颗粒，在4摄氏度下辅以蛋白酶抑制剂，轻轻混合10分钟。在孵育结束时，通过离心收集样品，将超级钠转移到预冷管中。在4摄氏度下加入100微升冰冷膜提取缓冲液，并辅以蛋白酶抑制剂，在4摄氏度下进行10分钟的孵育，轻轻混合。然后在4摄氏度和3000倍g下将颗粒离心5分钟，将超级钠收集到新的管中。为了收集可溶性核馏分，在4摄氏度下孵育样品30分钟之前，在颗粒中加入50微升的核萃取缓冲液，并辅以蛋白酶抑制剂。用涡流。在孵化结束时，将样品离心并收集中超。为了收集不溶性核馏分，加入50微升的核萃取缓冲液，并辅以蛋白酶抑制剂、氯化钙和微球蛋白酶，加入涡流颗粒。在37摄氏度下孵育样品5分钟，然后涡旋和离心。然后收集超自然。为了收集细胞骨骼分数，添加50微升的细胞骨骼提取缓冲液，并辅以蛋白酶抑制剂到含有涡流的颗粒中。在室温下孵育10分钟后，将样品离心并收集超钠。对于 SDS-PAGE 分析，在所收集的亚细胞分数样本的 20 微升中加入 7 微升 4X Laemmli 缓冲液，并在 100 摄氏度下将样品煮沸 5 分钟。在孵育结束时，将样品装载到丙烯酰胺凝胶上，并在120伏的恒定电压下执行电泳。然后将蛋白质以250毫安将蛋白质转移到硝基纤维素膜上90分钟。在庞塞奥染色溶液中孵育膜五分钟，以检查是否适当的蛋白质凝胶电泳和成功的印迹。在孵化结束时，用蒸馏水冲洗膜，直到背景清洁，并用Tris缓冲盐水连续清洗去除污渍，并在摇床上用Tween 20补充Tween 2010分钟。接下来，用阻尼溶液孵育膜，在室温下摇动一小时，然后用Tris缓冲盐水加Tween 20洗涤三次，每次洗涤五分钟。上次洗涤后，将膜与在四摄氏度下过夜阻断溶液的原抗体混合，然后用 Tris 缓冲盐水加 Tween 20 进行三次洗涤，持续五分钟。上次洗涤后，将膜与适当的马萝卜过氧化物酶结合的二次抗体混合在室温下阻断溶液中一小时，然后用Tris缓冲盐水加Tween 20洗涤膜，每次洗涤5分钟。上次洗涤后，使用化学发光检测蛋白带，并通过 ImageJ 量化西 Blot 波段的强度。在没有视网膜酸和脑源神经营养因子的情况下，未分化的SH-SY5Y细胞假设一个圆状的形态并形成细胞团。不出所料，经过五天的视网膜酸治疗后，团块会放松，细胞会扩散开来。在大脑衍生神经营养因子的另外三天治疗后，细胞通过分枝神经细胞网络出现均匀分布和互连。tau-NLS或tau-NES转染后，通过抗陶抗体的免疫荧光可以检测陶亚细胞定位。根据转染的效率，细胞在tau-NLS转染后与DAPI信号合并，或在tau-NES后与空核进行细胞质染色。Western Blot 分析揭示了细胞骨骼分数内的行为素、细胞质和细胞骨骼分数内的多巴蛋白和核分数内的 H2B 的富集。西布洛特分析还确认了核舱内标记和未标记 tau 的表达，车辆谷氨酸运输器在总提取物中只表达一个表达式。事实上，可溶性核和细胞质分中tau的比例突出了一个事实，即tau-NLS在可溶性核馏分中高度丰富，而tau-NES则减少。此外，tau-NLS 在细胞质分数的染色质含量中富含染色质含量，而tau-NES则减少。请务必仔细检查用于每个样本分馏的细胞数量，并验证在适当分数中内务基因的富集性。观看此视频后，您应该能够隔离细胞隔离室并评估 tau 在细胞核中的功能作用。

Research

JoVE Journal

Genetics

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

科研

遗传学

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Controllable Ion Channel Expression through Inducible Transient Transfection

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

qkat: 杀手细胞免疫球蛋白样受体基因的定量半自动分型

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...