Name: in vitro establishment of a genetically engineered murine head and neck cancer cell line using an adeno-associated virus-cas9 system
Uploaded: 2020-01-09T12:00:00
Description: Watch this Scientific Journal Video about In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System at JoVE.com

我们要感谢丹尼尔·吉特勒博士为我们提供了pAd Delta 5帮手质粒。这项工作由以色列科学基金会（ISF，700/16）（致ME）、美国-以色列两国科学基金会（BSF，2017323）（对ME和MS）、以色列癌症协会（ICA，20170024）（对ME）、以色列癌症研究基金会（ICRF，17-1693-RCDA）（对ME）和关注基金会（#7895）资助。研究金：阿隆奖学金，ME和BGU克雷特曼奖学金给SJ和MP。

这项研究得到了内盖夫动物护理和使用委员会的本古里安大学的批准。动物实验获得IACUC（IL.80-12-2015和IL.29-05-2018（E））的批准。本研究中使用的动物试验、住房和环境条件的所有方面都符合《实验室动物护理和使用指南》13。
1. 阿德诺相关病毒生产
<ol><li>第1天：细胞培养<ol>
<li>种子 4 x 106 HEK293T 细胞每 14.5 厘米板在 15 mL 的 DMEM.准备10个板，用于用...

<ol>
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J., 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159 (2), 440-455 (2014).</li><li>Zhang, Y., Chirmule, N., Gao, G. P., Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand-dependent+activation+of+cytotoxic+T+lymphocytes+by+adeno-associated+virus+vectors+in+vivo:+role+of+immature+dendritic+cells.">CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells.</a> Journal of Virology. 74 (17), 8003-8010 (2000).</li><li>Xiao, X., Li, J., Samulski, R. J. <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+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> Journal of Virology. 72 (3), 2224-2232 (1998).</li><li>Auricchio, A., Hildinger, M., O'Connor, E., Gao, G. P., Wilson, J. 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以前已经使用几种方法来转化培养中具有可变成功率的初级细胞25，26，27，28。这些方法大多使用小鼠成纤维细胞进行转化14、17、18、19或使用致癌物质，如4-硝基基苯丙胺-1-氧化物（4-NQO）21，22用于开发鼠细胞系模型。<sup...

HNC是一种常见的恶性肿瘤，全世界1.模拟HNC新奇的起源目前处于一个科学的转折点2。虽然在HNC2、3、4（例如TP53、PIK3CA、NOTCH1、FAT1和RAS）中发现了许多基因突变，但诱导HNC所需的基因组突变的具体组合仍不清楚。
目前人类HNC细胞系的使用大大有助于阐明与发病机制和治疗相关的机制3。然而，研究免疫功能低下的鼠系中的人类细胞系有其局限性，因为这些系统没有解决体内肿瘤过程、特定基因突变的作用以及免疫微环境中的治疗反应。因此，具有特定基因改变的鼠细胞系的发育和建立对于研究不同基因如何促进转化过程以及测试免疫能力小鼠的新型分子疗法至关重要。
生物医学研究中的基因功能研究受到DNA编辑技术的进步的显著影响，例如引入双链断裂（DSBs）。这些技术，包括使用锌指核酸酶、转录活化剂样效应核酸酶和聚类调节间隔短回触性重复（CRISPR/Cas9），允许在其位点操作任何感兴趣的基因。最新的CRISPR/Cas9系统由引导RNA（gRNA）组成，该RNA指示Cas9核酸酶在基因组中的特定位点生成DSB。该系统在修改任何细胞或目标组织的内源性基因方面得到了广泛的认可，即使在最传统的难以治疗的有机体5中也是如此。由于其简单性、速度和效率，它比其他系统具有多种优势。
在肿瘤学领域，CRISPR/CAS9技术满足了有效模仿癌细胞的需求。为了在HNC建立这个系统，我们操纵了有效的KRAS肿瘤基因和两个重要的肿瘤抑制基因，APC和p536。根据GENIE数据库7，这种组合在HNC是罕见的。RAS 突变（HRAS、NRAS 和 KRAS）仅在所有 HNC 人群中存在 +7%。这些肿瘤往往对治疗8，9具有抗药性。
Cas9及其gRNA的传递是通过使用AAV或慢病毒的病毒转导实现的。重组AAV通常是向目标细胞传递基因的首选方法，因为它的度子含量高，免疫反应温和，能够换出广泛的细胞范围，而且整体安全。使用AAV系统，已经生成了各种组织特异性小鼠细胞系，新的细胞系仍在开发中。然而，一个高效的基因组编辑系统，可以产生鼠HNC细胞系模型细胞仍有待开发。在这项研究中，我们寻求开发一种基于体外AAV-Cas9的系统，用于将原生鼠舌细胞转化为肿瘤状态。这种独特的CRISPR/Cas9转化方案和已建立的肿瘤细胞系可用于更好地了解由基因组变化引起的HNC生物学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-035-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-035-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>14697</td>
			<td></td>
		</tr>
		<tr>
			<td>Cre</td>
			<td>BioLegend</td>
			<td>900901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-AffiniPure Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-165-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy-AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-165-144</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9996</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2)</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal anti E cadherin</td>
			<td>Cell Signaling Technology</td>
			<td>3195S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal anti-KRT 14</td>
			<td>Abcam</td>
			<td>AB-ab181595</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946; actin</td>
			<td>MP Biomedicals</td>
			<td>691001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946; catenin</td>
			<td>Cell Signaling Technology</td>
			<td>9582S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK93T</td>
			<td>ATCC</td>
			<td><a href="https://www.atcc.org/en/Products/All/CRL-3216.aspx" target="_blank">CRL-3216</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Media, Chemicals and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Bio-Rad</td>
			<td>30015484</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Amresco</td>
			<td>0332-TAM-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL (Westar Supernova and Westar Nova 2.0)</td>
			<td>Cyanagen</td>
			<td>XLS3.0100 and XLS071.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>04-127-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin - Agarose</td>
			<td>Sigma</td>
			<td>H6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolate II Genomic DNA Kit</td>
			<td>Bioline</td>
			<td>BIO-52066</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Panreac AppliChem</td>
			<td>300283</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio Lab Ltd</td>
			<td>1903059100</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep Solution</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Santa Cruz Biotechnology</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor cocktail</td>
			<td>Biotool</td>
			<td>B15001A/B</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>MilliporeSigma</td>
			<td>P2714-1BTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer</td>
			<td>MERCK Millipore</td>
			<td>648311-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Thermo Fisher Scientific</td>
			<td>18047019</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>MilliporeSigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>03-050-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Bar Naor</td>
			<td>BNCB00130RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Bar Naor</td>
			<td>BN9308C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J</td>
			<td>Jackson labs</td>
			<td>24857</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid)</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR</td>
			<td>Broad Institute of MIT</td>
			<td></td>
			<td>Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA</td>
		</tr>
		<tr>
			<td>AAV 2/9 capsid vector</td>
			<td>Addgene</td>
			<td>112865</td>
			<td></td>
		</tr>
		<tr>
			<td>pAD Delta F5 helper</td>
			<td>Ben Gurion University of the Negev</td>
			<td></td>
			<td>Provided by Dr Daniel Gitler, Department of Physiology and Cell Biology, Faculty of Health Sciences, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.</td>
		</tr>
		<tr>
			<td>Plastic wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon-ULTRA filter 100 KDa</td>
			<td>Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m sterile filters, 4 mm</td>
			<td>Millex</td>
			<td>SLGV004SL</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m sterile filters, 13 mm</td>
			<td>Millex</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plates</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro establishment of a genetically engineered murine head and neck cancer cell line using an adeno-associated virus-cas9 system

使用原发正常上皮细胞，利用聚集调控间隔，在肿瘤基因和肿瘤抑制基因中引入特定突变，可以重新诱导细胞转化所需的基因组改变基于小鼠的短音重复（CRISPR）基因组编辑技术。这项技术使我们能够准确地模拟使用小鼠发生的人类癌症的基因变化。通过基因转化鼠原细胞，我们可以更好地研究癌症的发展、进展、治疗和诊断。在这项研究中，我们使用可诱导的Cas9小鼠舌上皮细胞，在体外使用腺相关病毒（AAV）进行基因组编辑。具体来说，通过改变正常舌上皮细胞中的KRAS、p53和APC，在体外产生了一个小鼠头颈部癌（HNC）细胞系，这是合成小鼠的肿瘤。这里介绍的方法详细介绍了如何生成具有特定基因组改变的HNC细胞系，并解释了它们是否适合预测合成小鼠的肿瘤进展。我们设想，这种有前途的方法将是翔实和有用的研究肿瘤生物学和HNC的治疗。

使用 AAV 系统转换正常的 Cas9 单元 图1提供了本研究中使用的AAV转基因质粒的详细矢量图。图 2概述了基于 AAV-Cas9 的系统的设计与工作。为了产生病毒颗粒，使用PEI转染方法用AAV转录载体和其他病毒包装载体转染HEK293T细胞。转染后，对含病毒细胞进行收集和分离，采用肝素-腺素纯化工艺，对病毒颗粒进行纯化和?...

Watch this Scientific Journal Video about In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System at JoVE.com

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

为了了解肿瘤，需要开发在头颈部癌症患者中发生特定基因变异的鼠模型。在这里，我们提出了一个方案，用于使用腺相关病毒-Cas9系统对原性鼠舌细胞进行体外转化，以产生具有特定基因组改变的小鼠HNC细胞系。

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

该协议使头部和颈部癌症模型的发展与特定的基因组操作，显著影响我们了解特定基因突变在肿瘤过程中的作用。这种技术的主要优点是细胞系的易用性可以从几乎任何器官发展。演示程序将是马努普拉萨德一个研究生从我的实验室。首先使用手术剪刀从六周大的雄性或雌性B6转基因小鼠的舌头收获。使用手术刀将组织切碎成非常小的碎片，并收集成含有4.5毫升RPMI纯介质的15毫升管，不含血清。将200微升三重酶混合物加入管中，在37摄氏度下孵育样品，每10分钟敲击10分钟，敲击管子，增强组织的酶分离。在孵育结束时，停止PBS中5%FBS的反应，并通过70微米网状滤网过滤细胞悬浮液，将细胞从较大的组织片段中分离。通过离心收集细胞，并在三毫升完整介质中重新悬浮颗粒。然后将细胞板在三毫升完整的介质每16毫米盘。将保留在过滤器顶部的细胞聚合转移到单独的 16 毫米培养盘上，并添加三毫升的完整介质。将两个盘子放在培养箱中，直到形成不同的细胞菌落。经过一周的培养，微观检查原细胞培养是否存在成纤维细胞污染，在37摄氏度的02%EDTA中用25%的三丁普辛治疗细胞一分钟，并去除任何成纤维细胞。培养物的第10天在02%EDTA中使用25%的三至五分钟，在37摄氏度下从细胞悬浮液或细胞聚集培养物中采集细胞，并在每井两毫升的介质中看到两次10至第五个原细胞，进入6井板的每个井中。第二天，每毫升用10倍转导10至12个病毒基因组，并在37摄氏度下孵育含有病毒颗粒的介质中的细胞48小时。在孵化结束时，每井用两毫升新鲜完整培养基代替培养超级细胞，将细胞返回细胞培养箱。经过两个星期的培养扩张，播种细胞进行验证和体内肿瘤生成实验。使用 AAV CAS9 载体，已确定每毫升病毒的 10 倍到 12 个病毒基因组，以便使用所证明的协议有效地将原发性冰毒细胞转化为肿瘤基因图谱。转导细胞表达克里、CAS9和绿色荧光蛋白。注射五倍10至第五原 AAV 转导图到 NOD SCID 小鼠的舌头、嘴唇和皮肤，导致这些动物的肿瘤形成不同于初级图的注射，不会诱发肿瘤。通过免疫荧光和西印，可以确认原发性舌头上皮细胞的肿瘤原变。通过DNA深度测序进行基因组分析，从转化的细胞中分离出，通过AAV CAS9系统证实了肿瘤蛋白53和APC基因的有效基因编辑和帧移位。此外，转化的舌细胞在野生C57黑色六只小鼠中有效形成肿瘤。对肿瘤细胞的免疫体化学分析揭示了E-Cadherin和Keratin14的细胞质表达，这两种是上皮组织标记。尝试此程序时，请始终记住，太多的切碎或太多的酶反应会导致细胞的生存能力降低，从而降低病毒的事务效率。使用这个程序，细胞系可以进行基因改造，从任何感兴趣的组织中获得癌细胞的致肿瘤和转移潜力的洞察。

Research

JoVE Journal

Cancer Research

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

科研

癌症研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

遗传工程小鼠模型的散发性结肠直肠癌

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...