这项工作得到了由韩国政府 (MSIP) (NRF-2014K1A4A7A01074642) 和韩国国家研究基金会 (NRF) 个人科学家支持计划 (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239) 资助的赠款。

1. 慢生产 注意: 根据国家卫生研究院 (NIH) 有关重组的研究指南, 对慢载体进行生产和操作。dna.用增强 YFP (SOD1WT-YFP 和 SOD1A4V-YFP) 标记的野生型和 A4V 突变体 SOD1 的质粒, 在金 et al 中描述。 11 两种基因融合产品均采用 PCR 引物对5和 #8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3 和 #8242; 5 和 #8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3 和 #8242; 插入 pTRIP 三角洲 U3 巨细胞病毒质...

<ol>
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I., 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=Disulfide+bond+mediates+aggregation,+toxicity,+and+ubiquitylation+of+familial+amyotrophic+lateral+sclerosis-linked+mutant+SOD1.">Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1.</a> J Biol Chem. 282 (38), 28087-28095 (2007).</li><li>Juneja, T., 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=Prognosis+in+familial+amyotrophic+lateral+sclerosis:+progression+and+survival+in+patients+with+glu100gly+and+ala4val+mutations+in+Cu,Zn+superoxide+dismutase.">Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase.</a> Neurology. 48 (1), 55-57 (1997).</li><li>Kaur, S. 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作者没有什么可透露的。

有两种主要的方法来生成稳定的细胞系。第一次需要几个星期, 需要对基因组整合的质粒 DNA 载体进行瞬时转染和抗性选择。第二个需要几个小时的时间, 通过使用慢, 使这一协议的有效表达的目标蛋白在多个细胞系有限的努力。行程-CMV 矢量19是一个持续使用的载体, 具有保守的转导效率和稳定的转基因表达。令我们吃惊的是, 生成的三单元格线显示的是突变 SOD1 的可见聚合, 而不?...

蛋白质聚集是一种生物过程, 错误蛋白可以在神经退行性疾病 (淀粉样变性) 中起到致病剂的作用。在理解细胞功能障碍中聚合体的作用, 以及在促进发现影响病理发病的新因素方面, 蛋白质聚集性是必不可少的。活体细胞荧光标记蛋白的可视化是一种强有力的方法, 可以帮助开发适用于高含量筛选 (HCS)1、2、3、4的测试。
肌萎缩侧索硬化 (als) 被认为是一种 proteopathic 的疾病, 由存在的错误蛋白的倾向聚集和积累在运动神经元的家族 als (fALS) 和零星 als (销售)5,6.20% fALS 病例的一个子集与胞浆抗氧化酶铜锌超氧化物歧化因子 1 (SOD1)7,8的基因编码中的显性突变相关。提出了这一基因衍生性功能障碍的几个潜在原因, 包括 SOD1 变种的结构和功能的变化, 如异常稳定性, 增加的展开率, 和倾向于聚合9, 10。值得注意的是, 由 ALS 连接的 SOD1 变种和野生型 (重量) SOD1 所共享的唯一经过验证和潜在毒性的属性是形成条块分割的蛋白质聚合体或蛋白质夹杂物的倾向11,12.错误突变 SOD1, 是持续 polyubiquitinated 和退化的泛素-蛋白酶体系统。因此, 对蛋白酶体活性的抑制导致了突变体 SOD1 聚集13,14, 形成了由可溶性组分组成的无定形结构, 可与可溶性突变体 SOD1 交换。在胞15中。特别是, SOD1 突变 A4V (丙氨酸在密码子4改为缬氨酸) 是最常见的 ALS 引起的突变, 并导致快速神经的平均生存时间少于2年后疾病发作16。生化, SOD1 A4V 有增加的倾向 monomerize, 聚合, 并形成淀粉样孔隙;它的孔状聚合体类似于其他与疾病相关的突变体的淀粉样孔, 如α-核和β-淀粉样蛋白17。为了研究 SOD1-aggregate 积累的动力学规律, 对可溶性和不溶性 SOD1 聚合形式的监测方法有待发展。
我们以前已经显示, 使用活细胞成像和 HEK-293 细胞瞬时转染荧光蛋白标记 SOD1, ALS 相关的突变损害 SOD1 二和聚集11。虽然瞬态表达系统可以提供有关短期基因过度过度的生物学结果的有用信息, 但提供所需基因的稳定整合的方法可能更适合于检测的发展。因此, 慢载体提供了赋予哺乳动物细胞长期和调控基因表达的能力18。在这项研究中, 我们的重点是生成稳定细胞系转与重组慢轴承和突变 SOD1 标记的黄色荧光蛋白 (YFP)。使用活细胞成像显微镜和自动定量的 SOD1 聚集, 我们触发和量化 SOD1 聚集事件时抑制蛋白酶体。

<table>
	<tbody>
		<tr>
			<td>ALLN (C20H37N3O4)</td>
			<td>Millipore</td>
			<td>208719</td>
			<td></td>
		</tr>
		<tr>
			<td>MG132 (C26H41N3O5)</td>
			<td>Sigma-Aldrich</td>
			<td>C2211</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxomicin (C28H50N4O7)</td>
			<td>Sigma-Aldrich</td>
			<td>E3652</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H-3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera</td>
			<td>Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera EvoShell software</td>
			<td>Perkin Elmer</td>
			<td>Ver 1.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Operetta</td>
			<td>Perkin Elmer</td>
			<td>OPRT1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmony Imaging software</td>
			<td>Perkin Elmer</td>
			<td>Ver 3.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Ver 2.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CyBi Hummingwell liquid handling</td>
			<td>CyBio AG</td>
			<td>OL 3387 3 0110</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

肌萎缩侧索硬化症 (ALS) 是一种致命的神经退行性疾病, 可导致遗传突变的基因编码铜锌超氧化物歧化酶 1 (SOD1)。SOD1 的结构不稳定性和家族性 als 患者的 SOD1-positive 包裹体的检测支持错误和/或聚合 SOD1 在 als 病理中的潜在因果作用。在这项研究中, 我们描述了一个细胞分析的发展, 旨在量化的动态 SOD1 聚集在活体细胞的高含量的筛选方法。利用慢向量, 我们生成了表达野生型和突变 A4V SOD1 标记的稳定细胞系, 黄色荧光蛋白, 发现这两种蛋白质都表达了胞没有任何聚集的迹象。有趣的是, 只有 SOD1 A4V 稳定表达在 HEK-293, 但不是在 U2OS 或 SH-SY5Y 细胞线, 形成了聚集后, 蛋白酶体抑制剂治疗。我们表明, 这是可能的量化聚合的基础上, 剂量反应分析的各种蛋白酶体抑制剂, 并跟踪集料形成动力学的延时显微镜。我们的方法引入了定量的可能性, ALS 突变的作用, SOD1 在聚合形成和筛选的小分子, 防止 SOD1 A4V 聚集。

使用慢生成稳定的单元格线:在图 1中说明了监测 SOD1 蛋白聚合体的总体策略。在第一步, 我们生成了一个慢表达载体, SOD1 稳定基因传递到细胞系 (步骤 1)。制备了两个慢矢量编码 YFP 标签 SOD1 和 SOD1 A4V (SOD1WT-YFP 和 SOD1A4V-YFP 分别) 与包装和包络质粒 (图 2A)。在慢转导 (步骤 2) 测试的细胞线 (HEK-293, U2OS 和 SH-SY5Y) 保持健?...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

我们描述一种方法来量化错误蛋白质的聚集。我们的协议细节慢诱导稳定细胞线生成, 自动共焦成像, 和图像分析的蛋白质集料。作为一个说明性应用, 我们研究了小分子在促进 SOD1 聚集的时间和剂量依赖性的方式的影响。

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.The implication of this technique extends towards potential therapy of neurodegenerative diseases, such as ALS, because it proposes a simple method that allow to develop cellular assay and perform screening for small molecule moderators. To start virus production, prior to plasmid transfection split HEK-293 T-cells at 30 to 40%confluence in a 10 centimeter tissue culture dish and 10 milliliters of cell culture medium. Incubate the culture plate in a 5%carbon dioxide incubator at 37 degrees Celsius overnight.On the following day, prepare DNA transfection solution with packaging plasmids lentiviral vector, 125 microliters of one molar calcium chloride and add distilled water to adjust the volume to 500 microliters. Gently add 500 microliters of 0.05 molar HEPS buffer and incubate for 10 minutes at room temperature. After that, replace HEK-293 T-cell medium with 10 milliliters pre-warmed antibiotic-free fresh culture medium mixed with one milliliter of DNA transfection solution.Incubate at 37 degrees Celsius and 5%carbon dioxide incubator over night. Replace cell supernatent with fresh culture medium on the following day and incubate again overnight. Finally, on the following day harvest the supernatent and transfer to a 15 milliliter tube.To remove the dead cells and debris, centrifuge the tube at 500 times g for five minutes. To further purify the supernatent, pass it through a 0.45 micrometer filter using a 60-milliliter syringe. Then, immediately dispense the filtered supernatent into 300-microliter single use aliquots.To prepare the cells for transduction, split them to be infected at 60%confluence in a six-well tissue culture plate in two milliliters of DMAM supplemented with 10%FBS. Incubate the cells in a 37-degree Celsius incubator at 5%carbon dioxide overnight. On the following day, thaw one aliquot of the frozen lentivirus on ice for each use.Meanwhile, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, dilute it in DMEM in new 1.5 milliliter micro-centrifuge tubes. Then adjust the volume to one milliliter with reduced serum medium.Next, add two microliters of polybrene to one milliliter of virus per medium and mix well by pipetting. Add one milliliter of this mixture to the cells and incubate at 37 degrees Celsius for 24 hours. After incubation, remove the virus containing medium and replace it with normal DMEM supplemented with 10%FBS and return the cells to the incubator.Monitor the cell growth and change the medium every two days When the cells reach confluence, expand each well of the six-well dish into a 10-centimeter tissue culture plate. Move the plates to the incubator. Finally, save 1.5 times 10 to the 4th cells per well and 50 microliters of DMEM on 384 well assay plates.Then check the expression level of the YFP-tagged protein under the microscope. If the expression shows signal-to-noise ratio equal or greater than three, prepare cell stock for the corresponding cell line. For the compound transfer, on a 384-well storage polypropylene plate, add serial dilutions of compounds by the standard one to three dilution series.Then, using a liquid handler equipped with a 384-capillary head, distribute 0.25 microliters of proteasome inhibitors on empty, flat-bottomed black 384-well assay plates. Then seed 1.5 times 10 to the 4th cells on these plates. Incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.Prior to imaging the cell line treated with proteasome inhibitors, add 10 microliters of pre-warmed DMEM with staining solution and incubate at room temperature for 10 minutes. After staining, start the automated microscope operating software by selecting the microscope tab. First, select the configuration tab and then select 20 times objective in the correct plate type.To allow proper focus with different plate types, make sure that the caller is set to the correct value on the objective. Next, select exposure one. Set focus height to zero micrometers, then select focus.Once it is focused, expose camera one. To optimize the exposure plane, adjust the focus height and click on take height. Change exposure times and laser power for a maximum pixel intensity of approximately 3000 and save exposure parameters.Select experiment definition tab, create a layout and sublayout. Next, drag and drop the relevant layout exposure, reference image, skew crop file, and sublayout. Then, save the experiment.Finally, select automatic experiment tab and acquire images. For two-channel images, first select the software algorithm to segment the primary objects, such as nuclei, cytosol, and aggregates. First, distribute 0.25 microliters of proteasome inhibitor dissolved in 100%DMSO or DMSO on 384-well flat-bottomed plates.On the same plate, manually seed 1.5 times 10 to the 4th cells per well in 50-microliters of DMEM. Then set up the microscope system environmental control unit to 37 degrees Celsius and 5%carbon dioxide. Operate the microscope in wide-field florescence mode and as software settings, choose LWD 20 times objective non-confocal mode, four fields per well, and capture interval of one hour.Then, select the appropriate algorithm to segment the primary object such as cytosol and aggregates. Adjust background threshold and contrast parameters if required. The tested cell lines transduced with SOD-1 wild type and SOD-1 A4V mutant remained healthy, showed high expression levels, and longterm wide phi-labeling irrespective of the SOD form.Treatment with proteasome inhibitor ALLN for 24 hours promoted the accumulation of SOD-1 A4V YFP aggregates in HEK-293 cells but not in SOD-1 Wild Type YFP transduced cells. Furthermore, there were very low levels of aggregation in U2 OS and SH-SY5Y cell lines SOD-1 A4V YFP transduced with the same vectors. After seeding the cells in the presence or absence of ALLN, they were monitored for aggregate formation for a period of 50 hours.ALLN aggregate formation reached a plateau at 24 hours and remained stable over the next 12 hours. Furthermore, cell density significantly decreased after 28 hours as a result of the cytotoxic effect of ALLN, while cell number increased in the DMSO-treated negative control. SOD-1 A4V YFP cells cultured in the presence of proteasome inhibitors for 24 hours were stained with Hoechst solution.The relative toxicity for three different proteasome inhibitors was quatified by measuring the number of adherent cell lines remaining in the well labeled with Hoechst dye. The total cell number decreased in response to increased dose of protease inhibitors. Inversely, the percentage of cells expressing SOD-1 A4V aggregates increased with the concentration of protease inhibitor.When doing this process it is important to remember that the cell quality will drastically influence your result. So it's important to determine that the cells are microplasma free and are healthy all along the steps. Following this process, another imaging method can be used in order to answer additional questions regarding the degradation of protein or the interaction of the protein with others using resonance energy transfer.After watching this video, you should have a good understanding of how to generate the tools to test the fate of protein in multiple living cells using high-content image analysis. So this method can provide insight into protein aggregation as it occurs in simple cellular models. It can also be applied to other systems such as neuron differentiated from patient specific induced pluripotent stem cells.

assay-development-for-high-content-quantification-sod1-mutant-protein

Research

JoVE Journal

Neuroscience

7.1K Views.  Institut Pasteur Korea. The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.The ...