Name: cell-cell fusion of genome edited cell lines for perturbation of cellular structure and function
Uploaded: 2019-12-07T17:00:00
Description: Watch this Scientific Journal Video about Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function at JoVE.com

这项工作由威尔康信托亨利·韦康奖学金资助R.M.（https://wellcome.ac.uk/grant号100090/12/Z）。在研究设计、数据收集和分析、决定出版或编写手稿方面，受资者没有作用。我们感谢阿肖克·文基塔拉曼和保罗·法兰西对该项目提出的重要建议和指导。我们感谢剑桥医学研究所流细胞测定设施中的基亚拉·科塞蒂和加布里埃拉·格龙迪斯-科塔巴的大力支持。我们感谢利亚姆·卡西迪、托马斯·米勒和吉安马科·孔蒂诺校对手稿。

1. 差分荧光细胞标签
<ol><li>使用 CRISPR Cas9 进行基因标记<ol>
<li>使用CRISPR Cas9基因组编辑将根蛋白（或其他感兴趣的基因）与人类癌细胞系中的荧光蛋白meGFP或mscarlet-I标记。 注：基因组编辑的详细方案在24、25、26日其他部分进行。</li>
	</ol>
</li>
	<li>荧光染料标签<ol>
<li>在Dulbeco的改性Eagle培养基（DMEM）中生长Cal51?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+glycol)+(PEG)-mediated+fusion+between+pure+lipid+bilayers:+a+mechanism+in+common+with+viral+fusion+and+secretory+vesicle+release?.">Poly(ethylene glycol) (PEG)-mediated fusion between pure lipid bilayers: a mechanism in common with viral fusion and secretory vesicle release?.</a> Molecular Membrane Biology. 16 (4), 279-296 (1999).</li><li>Pontecorvo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+mammalian+somatic+cell+hybrids+by+means+of+polyethylene+glycol+treatment.">Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment.</a> Somatic Cell Genetics. 1 (4), 397-400 (1975).</li><li>Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+giant+polynuclear+cell+formation+caused+by+HVJ+virus+from+Ehrlich's+ascites+tumor+cells.+I.+Microscopic+observation+of+giant+polynuclear+cell+formation.">Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells. I. Microscopic observation of giant polynuclear cell formation.</a> Experimental Cell Research. 26, 98-107 (1962).</li><li>Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-mediated+fusion+and+related+electrical+phenomena.">Electric field-mediated fusion and related electrical phenomena.</a> Biochimica et Biophysica Acta. 694 (3), 227-277 (1982).</li><li>Hu, 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=Fusion+of+cells+by+flipped+SNAREs.">Fusion of cells by flipped SNAREs.</a> Science. 300 (5626), 1745-1749 (2003).</li><li>Bahadori, A., Lund, A. R., Semsey, S., Oddershede, L. B., Bendix, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+fusion.">Mechanisms of cell fusion.</a> Nature. 253 (5488), 194-195 (1975).</li><li>Mahen, R., Venkitaraman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+centrosome+assembly.">Pattern formation in centrosome assembly.</a> Current Opinion in Cell Biology. 24 (1), 14-23 (2012).</li><li>Mahen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+centrosomal+roots+disentangle+to+allow+interphase+centriole+independence.">Stable centrosomal roots disentangle to allow interphase centriole independence.</a> PLoS Biology. 16 (4), e2003998 (2018).</li><li>Mahen, R., 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=Comparative+assessment+of+fluorescent+transgene+methods+for+quantitative+imaging+in+human+cells.">Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.</a> Molecular Biology of the Cell. 25 (22), 3610-3618 (2014).</li><li>Ran, F. 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我们演示了一种简单且经济高效的方案，用于融合细胞，并用显微镜可视化细胞混合的后续架构，从开始到结束大约需要两天时间。该协议的关键部分是通过细胞分拣富化熔融细胞（协议第3节），以及通过显微镜对熔融细胞进行仔细验证（协议第4节）。这些部分确保熔融细胞容易获得，是真正的异体。应坚持浓度和孵育时间。例如，当在较高浓度或较长的孵育时间下使用时，细胞染料在流式细?...

细胞结构的静时维持对生命至关重要。细胞具有特征形态、亚细胞细胞细胞数和内部生化组合物。了解这些基本特性是如何产生的，以及它们在疾病期间是如何出错的，这需要实验室工具来干扰它们。
细胞融合是两个或多个独立细胞的融合。细胞融合可能是真核生物1的出现的关键。在人体内，细胞融合是比较少见的，发生在受限发育环境和组织类型，如受精期间或形成肌肉，骨骼和胎盘2。该协议描述了细胞-细胞融合在组织培养细胞系的诱导与微分荧光标记的细胞器，作为一个工具，了解控制细胞结构和功能的机制。
体外诱导细胞融合是单克隆抗体3的产生中心，是生物研究和疾病治疗的重要工具。细胞融合还被用来问许多不同的基本细胞生物学问题，关于细胞周期支配4，非倍体5，6，细胞重新编程7，8，修复受损的神经元9，病毒增殖10，凋亡11，肿瘤发生12，细胞骨骼动力学13，和膜融合14，15。基于实验室的方法诱导细胞融合16、17、18、19通过物理合并两个双层细胞合并成一个脂质膜。细胞融合可以由电诱导18，基于病毒的方法17，热质加热20，转基因表达19，以及包括聚乙烯乙二醇（PEG）16、21、22在内的化学物质。
中心体是控制细胞形状、运动、极化和除23的微管组织中心。中生菌根是纤维结构，延伸自含有蛋白质根蛋白24的中源体（由基因CROCC编码）。我们最近使用细胞融合来了解在异体内与父母细胞24相比，中位位置和数量是如何变化的。使用这种方法的原理是跟踪在微分荧光标记的亲子细胞融合后，在异体细胞内的根源细胞，从而成像细胞融合和裂变。荧光标记的蛋白质根蛋白-meGFP或根蛋白-mscarlet-I通过基因组编辑在单独的细胞系中产生，然后通过PEG介导的细胞融合融合。我们描述了使用细胞染料（材料表）来识别融合细胞通过流式细胞学和随后的荧光显微镜鉴定的中源体细胞的起源和形态（图1）。这种方法是一种强大而独特的方法，用于研究细胞状态的重大变化，包括细胞细胞数量如何影响细胞平衡。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml tube</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>F8875</td>
			<td></td>
		</tr>
		<tr>
			<td>880 Laser Scanning Confocal Airyscan Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-well imaging dishes</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP alpaca GFP booster nanobody</td>
			<td>Chromotek</td>
			<td>gba-488</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Influx Cell Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Filters (70um)</td>
			<td>Biofil</td>
			<td>CSS010070</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>ThermoFisher Scientific</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>ThermoFisher Scientific</td>
			<td>C34571</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoTag-X2 anti-mScarlet-I alpaca nanobody</td>
			<td>NanoTag Biotechnologies</td>
			<td>N1302-At565</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 CO2 independent imaging medium</td>
			<td>Sigma-Aldrich</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red free DMEM, high glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>21063029</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1 x PBS)</td>
			<td></td>
			<td></td>
			<td>8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2HPO4, dH2O up to 1L</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol Hybri-Max 1450</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>BP151</td>
			<td>nonionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher BioReagents</td>
			<td>BP337</td>
			<td>nonionic detergent</td>
		</tr>
	</tbody>
</table>

cell-cell fusion of genome edited cell lines for perturbation of cellular structure and function

生命在脂质膜内的空间划分，允许细胞和细胞器内分离形成不同的分子状态。细胞融合是两个或多个细胞的合并，形成单个细胞。在这里，我们提供了两种不同细胞类型的细胞融合协议。融合混合细胞通过基于流式细胞学的分类进行浓缩，然后是混合细胞结构和功能的荧光显微镜。基因组编辑产生的荧光标记蛋白在融合细胞内成像，允许根据荧光发射识别细胞结构，并引用回源细胞类型。这种稳健和一般的方法可以应用于不同的细胞类型或感兴趣的细胞器，以了解细胞结构和功能跨越一系列的基本生物学问题。

在流式细胞测量过程中，荧光信号高于未标记的控制细胞（图2A）。盖茨被设置为对双阳性细胞进行分类，将这个群体直接浓缩成成像皿，以便进行进一步的微观分析。熔融细胞可检测为明显的双荧光阳性细胞，约占种群的±1%。
融合通过将两个细胞混合成一个（图<stron...

Watch this Scientific Journal Video about Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function at JoVE.com

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

该协议的目的是融合两种不同的细胞类型，以创建混合细胞。熔融细胞的荧光显微镜分析用于跟踪细胞细胞器来源细胞。此测定可用于探索细胞结构和功能如何对细胞融合对扰动的反应。

基因组编辑细胞系细胞融合，用于细胞结构和功能的扰动

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...

此方法允许细胞结构和功能通过两种不同细胞类型的融合来干扰，以解决细胞如何响应细胞器数量变化的问题。通过成像熔融细胞内的荧光标记蛋白质，细胞结构可以参考回细胞起源类型，以研究细胞平衡和功能的机制。对于荧光染料标签，请参阅感兴趣的人类癌细胞群，这样到第二天早上，每种培养体将至少扩大至六倍，达到第六细胞的70%至90%。在贴标当天，用每次洗涤 10 毫升 PBS 洗涤根素 eGFP 和根素-mScarlet 细胞两次。将紫光细胞染料在PBS中稀释至500纳米摩尔，在PBS中稀释至200纳米摩尔。用稀释的紫罗兰细胞染料和根莱特-mScarlet细胞在室温下用稀释的远红细胞染料标记根素eGFP细胞一分钟。在孵育结束时，停止与10毫升DMEM反应5分钟。用 10 毫升新鲜 PBS 清洗标记的细胞培养物和一个未标记的亲细胞培养物，然后用一毫升预谋的尝试性纤维素孵育所有培养物。在孵育结束时，将分离的细胞溶液转移到单个15毫升圆锥管中进行离心，并轻轻地将标记的染料颗粒重新在PBS的一毫升和未标记的细胞中，在一毫升DMEM中。然后将所有细胞样本返回细胞培养箱。对于细胞融合实验，将500微升紫罗兰染料标记细胞与500微升远红染料标记细胞混合到新的15毫升管中，通过离心沉淀剩余的未混合标记细胞。将颗粒重新放入一毫升新鲜DMEM中，将细胞放回培养箱中。通过离心收集混合细胞，并在30秒内以滴入颗粒中，以滴入700微升50%1450PEG。在室温下3.5分钟后，在30秒内加入10毫升无血清DMEM滴入，将管子放入培养箱10分钟。在孵化结束时，通过离心沉淀混合细胞，并轻轻地将颗粒重新悬浮在一毫升完整的介质中。为了通过 FACS 丰富熔融细胞群，通过 70 微米滤株轻轻地将所有细胞过滤到单个 FACS 管中，并在 Cytometer 软件中创建正向散射与侧散射和双体分块。运行未标记的细胞以记录其荧光强度，并创建用于识别熔融细胞的门。接下来，运行单正紫红色染料标记细胞，以确认紫色信号没有溢出到远红色通道和单正远红染料标记细胞，以确认紫色信道中没有远红色信号。然后短暂运行融合样本，以确认融合的细胞是否与这些门一起可见。设置分拣参数后，将分拣流集中对齐到包含 100 微升生长介质的八井成像盘中，并直接将双正融合细胞分类到培养皿中。当所有细胞都已分类后，立即将培养皿放入细胞培养箱中两到 16 小时。对于免疫荧光染色，首先用200微升4%的半氟醛替换细胞培养上流剂，在室温下孵育15分钟。在固定结束时，每次洗涤用200微升PBS洗三次细胞，然后将细胞渗透在200微升0.1%非日象剂中，在室温下10分钟。接下来，在室温下用200微升3%牛血清白蛋白进行非特异性结合，在室温下30分钟，然后用150微升染色缓冲液中的抗体进行标记。在室温下一小时后，在300微升PBS中清洗细胞两次，每次洗涤5分钟。上次洗涤后，用 200 微升新鲜 PBS 更换洗涤液。要获取融合细胞的图像，请使用能够进行四色成像的适当荧光显微镜并收集三维图像。在流式细胞测量过程中，荧光信号在比未标记的控制细胞水平更高时可见适当标记的细胞。门可以设置为排序，以丰富双阳性细胞群体直接到成像盘进一步微观分析。融合通过将两个细胞混合成一个细胞，诱导细胞结构的主要重新排列。异核子被确定为包含两个荧光染料信号的细胞，在单个细胞内混合，而不干预血浆膜。此外，在光明场成像的融合细胞中，也可以看到两个核。细胞结构和功能可以通过合并含有mGFP和mScarlet标记蛋白的细胞来进一步研究。当分体四环成分NEDD1被荧光标记时，融合会导致异核体内可见的百分体数增加一倍，至少四分之一的半环物质。细胞与内源性荧光标记根蛋白的融合允许在异核生物中识别每个中心体的起源细胞。该协议演示了如何融合微分标记的细胞系，以及如何对细胞器进行成像，以了解其结构和功能。与类似方法相比，这种健壮的协议相对容易，执行成本也相对低廉。这种技术可以用来提出一系列问题，例如细胞器融合是如何调节的，以及细胞对亚细胞细胞器数量的变化的反应。

Research

JoVE Journal

Developmental Biology

Method of Studying Palatal Fusion using Static Organ Culture

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

科研

发育生物学

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

建立基因组编辑的人多能干细胞系：从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...