Name: efficient recombinant parvovirus production with the help of adenovirus-derived systems
Uploaded: 2012-04-23T12:00:00
Description: Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

我们感谢团队Bioccelerator的病毒生产和开发单位，特别是马库斯·米勒，西尔维娅·明斯特曼，芭芭拉Liebetrau，曼迪罗雪尔。这项研究已部分由联邦教育和研究部（BMBF）和亥姆霍兹联合会在德国Krebsforschungszentrum /杜大CancéropôleEST在应用肿瘤病毒学联合计划的框架内部赠款支持。

请注意，与生物安全2级实验室重组细小病毒的（recPV）生产的需要。 该协议是在两个主要部分细分。第一部分通过转）（recPV生产需要作为接种服务的协议的第二部分（通过感染recPVs生产）生产最小recPVs的金额。一旦产生少量的recPVs，该协议的第一部分可以省略，只能通过提供感染细小病毒衣壳蛋白通过腺病毒辅助（见下文）的编码基因扩增和重组细小病毒。 <p class="jove_t...

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	<li>Cotmore, S. F., Tattersall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvoviral+host+range+and+cell+entry+mechanisms.">Parvoviral host range and cell entry mechanisms.</a> Adv. Virus. Res. 70, 183 (2007).</li><li>Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+parvoviruses+as+cancer+therapeutics.">Oncolytic parvoviruses as cancer therapeutics.</a> Cytokine Growth Factor Rev. 21, 185 (2010).</li><li>Hristov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+Its+Nonstructural+Protein+NS1,+Parvovirus+H-1+Induces+Apoptosis+via+Accumulation+of+Reactive+Oxygen+Species.">Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species.</a> J. Virol. 84, 5909-5909 (2010).</li><li>Cornelis, J. J., Salome, N., Dinsart, C., Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+based+on+autonomous+parvoviruses:+novel+tools+to+treat+cancer.">Vectors based on autonomous parvoviruses: novel tools to treat cancer.</a> J. Virol. 6, 193-193 (2004).</li><li>El-Andaloussi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adenovirus-based+helper+system+to+support+production+of+recombinant+parvovirus.">Novel adenovirus-based helper system to support production of recombinant parvovirus.</a> Cancer Gene Ther. 18, (2011).</li><li>Kestler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis+requirements+for+the+efficient+production+of+recombinant+DNA+vectors+based+on+autonomous+parvoviruses.">cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.</a> Hum. Gene. Ther. 10, 1619-1619 (1999).</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> J. Virol. 72, 2224 (1998).</li><li>He, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+system+for+generating+recombinant+adenoviruses.">A simplified system for generating recombinant adenoviruses.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 2509 (1998).</li><li>Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+purification+using+novel+methods+improves+infectious+titer+and+yield.">Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.</a> Gene. Ther. 6, 973 (1999).</li><li>Maxwell, F., Harrison, G. S., Maxwell, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+production+of+recombinant+AAV+by+transient+transfection+of+NB324K+cells+using+electroporation.">Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation.</a> J. Virol. Methods. 63, 129 (1997).</li><li>Wrzesinski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+and+pseudotyped+parvoviruses+minimize+the+contamination+of+recombinant+stocks+with+replication-competent+viruses+and+identify+a+DNA+sequence+that+restricts+parvovirus+H-1+in+mouse+cells.">Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.</a> J. Virol. 77, 3851 (2003).</li></ol>

我们已经表明，recPV生产，可以增强腺病毒基因组元素的存在。我们已增加超过10倍（从0.3到5 TU /细胞），通过转染腺病毒基因组元素提供超过100倍的合作感染与recPV结合的细胞与AD-VP辅助recPV产量比较传统的协议。这里所描述的协议可以进一步优化，确定交付的腺病毒基因组元素和被感染的AD-VP帮手的最佳浓度为最适当的时机。 较大的转基因（超过1600个碱基）RecPVs是生产效率?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td>西格玛</td><td> D5796 </td></tr><tr><td>的MEM </td><td>西格玛</td><td> M4655 </td></tr><tr><td>胎儿牛血清</td><td>临时机场管理局</td><td> A15-101 </td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030-024 </td></tr><tr><td> fugene </td><td>罗氏公司</td><td> 047097050001 </td></tr><tr><td>胰蛋白酶EDTA </td><td> Invitrogen公司</td><td> 25300062 </td></tr><tr><td> benzonase核酸</td><td>西格玛</td><td> E8263 </td></tr><tr><td>腺病毒-X的快速滴度套件</td><td> Clontech公司</td><td> 632250 </td></tr></tbody></table>

efficient recombinant parvovirus production with the help of adenovirus-derived systems

如鼠的H-1PV和MVM的啮齿动物细小病毒（PV），是小二十面体，单链DNA病毒。其基因组包含两个启动子P4和P38调节的非结构性（NS1和NS2）和衣壳蛋白（VP1和VP2），分别为1的表达。他们作为抗癌药物的溶瘤和oncosuppressive能力，而人类2非致病性高利率吸引。 NS1蛋白是该病毒的细胞毒性3的主要效应。为了进一步提高他们的天然抗癌活动，从这些载体的衍生工具已产生取代衣壳蛋白编码基因与治疗基因（ 如细胞毒性多肽，细胞因子，趋化因子，抑癌基因等）4。 （recPVs）重组细小病毒载体保留的NS1 / 2编码序列和光伏病毒DNA扩增和包装所必需的基因组端粒。生产recPVs只发生在生产者细胞（一般染HEK293T），合作的第二个表达载体为VP蛋白编码基因（ 图1）4（PCMV-VP）转染细胞。以这种方式产生的recPV载体是复制缺陷。 ，虽然recPVs证明拥有与尊重父母，他们已产生病毒增强oncotoxic活动，其生产仍然是一项重大的挑战和强烈阻碍了这些药物的使用在抗癌的临床应用。 我们发现，引进含有HEK293T细胞，E2A，E4（ORF6）和VA RNA基因（ 如 pXX6，质粒）提高超过10倍相比，在使用中的其他协议的recPVs生产AD-5派生载体。基于这一发现，我们已经构建一种新型的广告副总裁的助手包含腺病毒基因组元素以及必要加强recPVs生产parvovir我们VP基因单位5。 AD-VP辅助使用，允许生产REC-PVS使用协议，完全依赖于病毒感染步骤（如反对质粒转染），使人们有可能使用难以转染的细胞株（ 如 NB324K）（ 图2）。我们提出了一个方法，大大提高了重组病毒的产生量，降低生产时间和成本，不影响最终产品的质量5。此外，大规模生产的recPV（悬浮细胞和生物反应器）现在是可以想象的。

Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

在这里，我们描述只对细胞的感染，从而提高重组细小的生产效率超过100倍相比，在使用中的其他协议为基础的协议。此协议依赖于使用一种新型腺病毒含有的细小副总裁转录单位（AD-VP）5，基于辅助。

重组细小病毒与腺病毒派生系统的帮助下高效生产

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Research

JoVE Journal

Immunology and Infection

Estimating Virus Production Rates in Aquatic Systems

估计在水生系统的病毒产率

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...