Name: a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells
Uploaded: 2013-06-19T13:00:00
Description: Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

笔者想承认迈克尔·周，戴冰冰，梁萧此稿件提供数据。我们也承认约翰·格雷博士在这项研究中所使用的试剂厚礼。 PB是由国立癌症中心博士后奖学金支持。这项研究是由来自美国国立卫生研究院（R01AI68978 P01CA132681 RCA170820A），比尔和梅林达·盖茨基金会，平移加速转化医学联合中心和加州艾滋病毒/艾滋病的资助赠款的资助拨款研究计划。

DC-LV稳定生产细胞构造基于对GPR包装细胞系11包含必要的慢病毒组件gagpol，转和TET关闭系统。首先，逆转录病毒转导是用来产生一个GPRS编码一个四面体依赖SVGmu的糖蛋白的包装细胞系。然后，连环体数组转染用于转染的GPRS慢病毒载体的转基因如GFP细胞系。指定为LV-mGFP的这种稳定的生产细胞系，可在体外和体内进行测试，它能够产生一个DC-LV疫苗的GFP。 <p class="jove_tit...

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在这里，我们已经提出了一个方法生产大量使用慢病毒载体293T细胞稳定转关监管制度下TET所有慢病毒组件。至目前为止，大多数协议产生慢病毒载体依赖于标准的磷酸钙瞬时转染（见18）。这种方法已成功地在一个临床范围，但它可能遭受的一些限制，不太可能是存在于扩大生产的稳定细胞株。例如，与大量的LV质粒的共转染理论上可以增加复制能力的慢病毒19的发展机会。此外?...

已经开发了许多载体系统的基因传递。基于慢病毒载体已跻身研究最常用的病毒系统。这些载体是有利的，因为他们可以高效转染分裂和非分裂细胞1，由于整合到宿主基因组实现长期表达，表现出低的天然抗载体在大多数人群的免疫力，并具有低潜力从插入突变3,4的遗传毒性。 慢病毒的生产安全问题一直着色。慢病毒载体一般是来自于HIV-1，艾滋病的病原体。单个元件的慢病毒（转让，外壳和包装质粒）的瞬时转染是一种常见的和灵活的方法，在实验室设置中提供的遗传物质。然而，扩大规模的临床应用瞬时转染是繁琐和马的Ÿ导致复制能力的慢病毒5,6的发展。要克服这些障碍，稳定的包装和生产细胞系已开发6-11。这些线路之一，探地雷达的包装生产线11，受四环素有吸引力的优势。在本文中，我们将演示如何适应这种系统产生自我失活的慢病毒载体，是专门面向树突状细胞（DC-LVS）12。 树突状细胞（DC）是最强大的抗原呈递细胞的免疫系统。他们已经在癌症疫苗开发的主题极大的兴趣，因为它们直接启动，程序和规范肿瘤特异性免疫反应13。装有包括区议会有可能引出更强的抗肿瘤免疫反应比肽或DNA疫苗的接种方案。最近，我们已经开发出一种慢病毒载体，specificallÝ针对树突状细胞，通过修改辛德毕斯病毒糖蛋白，SVGmu 14。这些载体是独特的，他们表现出高特异性树突状细胞，并产生更强的免疫反应比特异性，VSVG假型载体。 在这里，我们报告的方法产生大量的这些直流针对性的慢病毒载体。我们表明，这些DC-LVS可以感染区议会和产生强烈的CD8 + T细胞的免疫反应。所有程序涉及动物的人道待遇，执行下USC Intitutional动物护理和使用委员会批准。为了执行在体内和临床实验，这是至关重要的建立的细胞系，可以制作高滴度的病毒。执行转导和转染与所述的相同的步骤，将产生这样的克隆的机会最大化。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

慢病毒载体（逻辑卷）是许多类型的细胞提供遗传材料的有力手段。由于相关的安全问题与这些HIV-1的衍生载体，产生大量的LVS是具有挑战性的。在本文中，我们报道了一种用于生产高滴度自我失活LVS的。逆转录病毒转导的tet过稳定线GPR生产细胞产生的细胞系，GPRS，它可以表示所有的病毒组分，包括树突状细胞特异性糖蛋白，SVGmu。然后，我们使用concatemeric的LV的转移质粒编码DNA转染，转染报道基因GFP的组合用一个可选择的标记。得到的克隆可以制作LV上面滴度大于10倍的我们实现与瞬时转染的。另外，这些病毒高效转染树突状细胞在体外和产生强烈的T细胞免疫反应向本报记者抗原。这种方法可能是一个不错的选择FOŗ强LV为基础的疫苗，癌症或传染病的临床研究。

在该方法中所述的稳定的细胞系，可以专门针对树突状细胞的慢病毒载体，大批量地生产。 图1B中所示，分离单个克隆，得到的稳定细胞株的不同的质量12。在测试的26个克隆中，有8产生的慢病毒颗粒滴度大于10 6转导单位每毫升（TU /毫升），这是一个典型的为SVG的假型化慢病毒载体瞬时转染产生的基准。在同一时间，几个克隆产生小于10 4 TU /毫升，隔离是很?...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

这里，我们用逆转录病毒转导和concatemeric的转染建立的细胞系中能表达的慢病毒载体（LV）的组件在没有四环素。此LV编码绿色荧光蛋白是一种糖蛋白，SVGmu，这是具体的树突状细胞上的受体与假。

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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