Name: an in vitro approach to study mitochondrial dysfunction: a cybrid model
Uploaded: 2022-03-09T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model at JoVE.com

这项研究是在由马里亚尼基金会资助的线粒体儿科疾病研究中心（http://www.mitopedia.org）进行的。VT是欧洲罕见神经肌肉疾病参考网络（ERN EURO-NMD）的成员。

注意：使用人类成纤维细胞可能需要道德批准。本研究中使用的成纤维细胞来自MD患者，并按照伦理要求储存在机构生物库中。对细胞的使用提供了知情同意。在室温（RT，22-25°C）的无菌层流柜下执行所有细胞培养程序。使用适用于细胞培养和无菌设备的无菌过滤溶液。在37°C的加湿培养箱中用5%CO2生长所有细胞系。应每周进行一次支原体检测，以确保无支原体培养。143BTK-rho0的细...

<ol>
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与nDNA相比，mtDNA具有非常高的突变率，因为缺乏保护性组蛋白并且其位置靠近呼吸链，这使得分子暴露于修复系统16无法有效抵消的破坏性氧化作用。第一个致病性mtDNA突变是在1988年发现的17，18，从那时起，已经描述了大量的突变。NGS技术是筛选整个线粒体基因组并轻松识别变异14的相关方法。然而，评估从未描述过...

线粒体疾病（MD）是一组多系统综合征，由核（nDNA）或线粒体（mtDNA）DNA 1突变引起的线粒体功能受损引起。它们是最常见的遗传性代谢疾病之一，患病率为1：5，000。mtDNA相关疾病遵循线粒体遗传学的规则：母体遗传，异质和阈值效应以及有丝分裂分离2。人类mtDNA是一个16.6 kb的双链DNA环，其中包含一个短的对照区，其中包含复制和转录所需的序列，13个蛋白质编码基因（呼吸链的所有亚基），22个tRNA和2个rRNA基因3。
在健康个体中，存在一个单一的mtDNA基因型（同质），而在病理条件下存在多个基因型（异质体）。有害的异质突变必须克服一个关键阈值，以破坏OXPHOS并引起可能影响任何器官的疾病，在任何年龄4。OXPHOS的双重遗传学决定了遗传：常染色体隐性遗传或显性遗传，nDNA突变X连锁，母体mtDNA突变，以及nDNA和mtDNA的散发病例。
在线粒体医学时代开始时，King和Attardi5 的一项具有里程碑意义的实验通过创建含有肿瘤细胞系细胞核的杂交细胞，其中mtDNA完全耗尽（rho0 细胞）和来自MD患者的线粒体，为理解导致MD的突变的起源奠定了基础。 并且确定核或线粒体基因组中是否存在突变并不容易。该方法于1989年描述，然后被在线粒体医学领域的几位研究人员使用6，7，8，9;最近发布了10个详细的协议，但尚未制作视频。如今，当NGS可以精确而快速地识别突变的位置时，为什么这样的协议应该具有相关性？答案是，cybrid生成仍然是最先进的方案，可以了解任何新型mtDNA突变的致病作用，将异质体的百分比与疾病的严重程度相关联，并在没有患者自身核背景贡献的同质核系统中进行生化研究11，12，13.
该协议描述了如何从在35mm培养皿中生长的融合的，患者来源的成纤维细胞中获得细胞形成体。在细胞查拉辛B存在下离心培养皿允许分离去核细胞体，然后在聚乙二醇（PEG）存在下与rho0 细胞融合。然后将得到的圆孢子在选择性培养基中培养，直到克隆出现。代表性结果部分显示了所得cybrids的分子表征示例，以证明mtDNA与供体患者的成纤维细胞相同，并且nDNA与肿瘤rho0 细胞系的核DNA相同。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-Bromo-2&#39;-Deoxyuridine</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>B5002-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Plates</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood and Cell Culture DNA extraction kit</td>
			<td>QIAGEN</td>
			<td>13323</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti J-25</td>
			<td>7,200 rcf, 37 &#176;C</td>
		</tr>
		<tr>
			<td>Centrifuge bottles, 250 mL</td>
			<td>Beckman Coulter</td>
			<td>356011</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin B from Drechslera dematioidea</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>C2743-200UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialyzed FBS</td>
			<td>Gibco</td>
			<td>26400-036 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate)</td>
			<td>EuroClone</td>
			<td>ECB7501L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline - PBS (w/o Calcium w/o Magnesium)</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Absolute Anhydrous</td>
			<td>Carlo Erba</td>
			<td>414601</td>
			<td></td>
		</tr>
		<tr>
			<td>FetalClone III (Bovine Serum Product)</td>
			<td>Cytiva - HyClone Laboratories</td>
			<td>SH30109.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes</td>
			<td>VWR</td>
			<td>M4150NO250SP4</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Research Microscope For Live Cell Microscopy</td>
			<td>Nikon</td>
			<td>ECLIPSE TE200</td>
			<td></td>
		</tr>
		<tr>
			<td>JA-14 Fixed-Angle Aluminum Rotor</td>
			<td>Beckman Coulter</td>
			<td>339247</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory autoclave Vapormatic 770</td>
			<td>Labotech</td>
			<td>29960014</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>EuroClone</td>
			<td>ECB 3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium MEM</td>
			<td>Euroclone</td>
			<td>ECB2071L</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-318</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG (Polyethylene glicol solution)</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>P7181-5X5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (solution 100x)</td>
			<td>EuroClone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Primo TC Dishes 100 mm</td>
			<td>EuroClone</td>
			<td>ET2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primo TC Dishes 35 mm</td>
			<td>EuroClone</td>
			<td>ET2035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100 mM</td>
			<td>EuroClone</td>
			<td>ECM0542D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5% in HBSS</td>
			<td>EuroClone</td>
			<td>ECB3051D</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>U3003-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro approach to study mitochondrial dysfunction: a cybrid model

进行氧化磷酸化（OXPHOS）的线粒体呼吸链复合物的缺乏是人类线粒体疾病的生化标志物。从遗传学的角度来看，OXPHOS代表了一个独特的例子，因为它是由两个不同的遗传系统互补产生的：核DNA（nDNA）和线粒体DNA（mtDNA）。因此，OXPHOS缺陷可能是由于影响核和线粒体编码基因的突变引起的。
King和Attardi于1989年发表的开创性工作表明，耗尽mtDNA（名为rho0）的人类细胞系可以被外源线粒体重新填充，以获得所谓的“透射粒体cybrids”。由于这些含有来自线粒体疾病（MD）患者的线粒体和来自rho0 细胞的细胞核的cybrids，因此可以验证缺陷是mtDNA相关还是nDNA相关。这些cybrids也是验证突变的致病性并在生化水平上研究其影响的强大工具。本文提出了一个详细的方案，描述了cybrid的产生，选择和表征。

产生cybrids需要3天的实验室工作加上一个选择期（约2周）和额外的1-2周用于克隆的生长。关键步骤是细胞质细胞的质量和选择期。环杂蚴的形态类似于rho0 供体细胞的形态。在环杂交中分配正确的mtDNA和nDNA是确认细胞身份所必需的。 图 2 给出了一个示例。在这种情况下，我们从来自携带异质m.3243A>G的患者的成纤维细胞开始产生cybrids，这是与线粒体肌病，脑病，乳酸?...

Watch this Scientific Journal Video about An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model at JoVE.com

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

透软骨圆柱体是通过将线粒体 DNA （mtDNA） 耗尽的细胞 （rho0 细胞） 与来自线粒体疾病患者的细胞形成体（去核细胞）融合而获得的杂交细胞。它们可以确定疾病的核或线粒体起源，评估生化活性，并确认mtDNA相关变异的致病作用。

研究线粒体功能障碍的 体外 方法：一种Cybrid模型

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human ...

网络生成仍然是了解任何新型线粒体DNA突变的致病作用并将三质体的百分比与疾病严重程度相关联的最新协议。该技术允许在均匀的核系统中进行生化研究，其中不存在患者核背景的贡献。该技术可以验证线粒体DNA突变的致病性。并使得有可能在生化水平上研究其影响 使用两毫升1X PBS（不含钙和镁）将含有成纤维细胞的35毫米培养皿清洗两次。用70%乙醇清洁盘子的外表面，等到酒精蒸发。从盘子上取下盖子，从瓶子上取下螺旋盖。将每个不带盖子的培养皿倒置在每个250毫升离心机瓶的底部。在每个瓶子中缓慢加入32毫升成核培养基，使培养基进入培养皿并与细胞接触。使用长玻璃牧场移液器去除盘子上的任何气泡，在本生火中弯曲尖端。用螺旋盖关闭每个瓶子，然后将其转移到离心机中。在37摄氏度和8， 000 x G下离心20分钟，从瓶子中吸出培养基并丢弃。通过将瓶子倒置在先前喷有70%乙醇的无菌纱布上来去除培养皿。用70%乙醇清洁培养皿的外表面及其盖子，并在乙醇蒸发后关闭培养皿。在继续之前，使用倒置显微镜检查活细胞的细胞形成。寻找极细长的成纤维细胞，因为它们的细胞核被细胞查拉辛诱导挤出。向每个35毫米培养皿中加入1， 000， 143行零细胞。重新悬浮在两毫升补充了5%FBS的培养基中。将培养皿在加湿的二氧化碳培养箱中放置三个小时，以将143排零细胞沉降在鬼魂上。孵育三小时后，吸出并丢弃培养基。用两毫升不含血清或MEM的DMEM高糖培养基洗涤粘附细胞两次，然后吸出并丢弃培养基。向细胞中加入500微升聚乙二醇溶液，孵育一分钟。吸出并丢弃聚乙二醇溶液。用两毫升DMEM高糖洗涤细胞三次，不含血清或MEM。加入两毫升熔融培养基，在37摄氏度的培养箱中用5%二氧化碳孵育过夜。胰蛋白酶消化剩余培养皿中的细胞计数并将它们接种到一个或多个培养皿中，在补充培养物中每个培养皿中加入50至100个细胞，直到出现克隆。然后让它们生长几天。使用体视显微镜，用克隆圆柱体或移液器尖端从培养皿中取出克隆。尽量避免池化不同的克隆并将其转移到96孔板中，每个孔含有200微升补充培养基。Cybrids是从来自携带异质M2343A2G的患者的成纤维细胞开始产生的，这是与线粒体肌病，脑病乳酸性酸中毒和中风样发作相关的最常见的线粒体DNA突变之一。对可变数量的串联重复的分析表明，网络DNA与行零细胞相同，证实了用143 B基因组替换了患者的网络DNA。限制性片段长度多态性或测序分析可用于评估线粒体DNA突变的存在和异质体百分比。当尝试此方案时，重要的是要验证核子和线粒体DNA的正确遗传资产，以及再填充的cybrids中的线粒体DNA量。

Research

JoVE Journal

Genetics

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

科研

遗传学

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

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 ...

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 ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

研究线粒体功能障碍的 体外 方法：一种Cybrid模型

研究线粒体功能障碍的体外方法：一种Cybrid模型