Name: engineering and evolution of synthetic adeno-associated virus (aav) gene therapy vectors via dna family shuffling
Uploaded: 2012-04-02T12:00:00
Description: Watch this Scientific Journal Video about Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling at JoVE.com

感谢作者在海德堡大学卓越CellNetworks的集群Chica和海因茨·夏勒博士（CHS）的基础以及出色的支持，他们的实验室，团队成员和工作。我们对此表示赞赏腺相关病毒的分子进化，通过DNA家族改组已成为一个非常活跃的领域，我们三年前首次出版以来，因此不能引用在这里由于篇幅所限，其工作的有关出版物的所有作者道歉。

1。制备质粒AAV衣壳基因编码集<ol><li>为了方便各种AAV衣壳基因 ​​（ 帽 ），随后的DNA改组的足够数量的常规准备，初步亚克隆质粒骨干到一个共同的基因。包括后来作为引物结合的巢式PCR和克隆网站（ 图2）使用相同的侧翼&gt; 20个核苷酸序列，这是非常重要的。 </li><li>使用适当的引物（见AAV5模范引物表），从常见的腺相关病毒载体通常包含AAV2 rep基因的选择帽</e...

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Gene Ther. 3, 281-304 (2003).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+methods+for+gene+transfer+vectors+based+on+adeno-associated+virus+serotypes.">Production methods for gene transfer vectors based on adeno-associated virus serotypes.</a> Methods. 28, 146-157 (2002).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+gene+therapy+vector+evolution+via+multispecies+interbreeding+and+retargeting+of+adeno-associated+viruses.">In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.</a> J. Virol. 82, 5887-5911 (2008).</li><li>Muller, O. 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W., Guy, J., Muzyczka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"humanized"+green+fluorescent+protein+cDNA+adapted+for+high-level+expression+in+mammalian+cells.">A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.</a> J. Virol. 70, 4646-4654 (1996).</li><li>Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+the+adeno-associated+virus+type+2+(AAV-2)+capsid:+epitope+mapping+and+identification+of+capsid+domains+involved+in+AAV-2-cell+interaction+and+neutralization+of+AAV-2+infection.">Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.</a> J. Virol. 74, 9281-9293 (2000).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatality+in+mice+due+to+oversaturation+of+cellular+microRNA/short+hairpin+RNA+pathways.">Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.</a> Nature. 441, 537-541 (2006).</li><li>Nakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unrestricted+hepatocyte+transduction+with+adeno-associated+virus+serotype+8+vectors+in+mice.">Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice.</a> J. Virol. 79, 214-224 (2005).</li><li>Koerber, J. T., Jang, J. H., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+shuffling+of+adeno-associated+virus+yields+functionally+diverse+viral+progeny.">DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.</a> Mol. Ther. 16, 1703-1709 (2008).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+selection+of+shuffled+AAV+genomes:+a+new+strategy+for+producing+targeted+biological+nanoparticles.">Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.</a> Mol. Ther. 16, 1252-1260 (2008).</li><li>Ward, P., Walsh, C. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotypes:+vector+toolkit+for+human+gene+therapy.">Adeno-associated virus serotypes: vector toolkit for human gene therapy.</a> Mol. Ther. 14, 316-327 (2006).</li><li>Kwon, I., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+gene+delivery+vectors:+molecular+engineering+and+evolution+of+adeno-associated+viral+vectors+for+enhanced+gene+transfer.">Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer.</a> Pharm. 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在这里，我们提出必要的实验步骤和AAV衣壳工程的指导方针，通过DNA家族改组，并在细胞或动物的进化。从本质上讲，这些协议是标准化的程序，我们的腺相关病毒领域内首次报道于2008年3版本。虽然一阵别人的后续研究已经报道了许多修改，例如 ，10-13，我们目前的版本代表产生重复性的结果，而服从缩放和适应任何需要的基本策略。 尽管我们的努?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DNA酶I </td><td> Invitrogen公司</td><td> 18068-015 </td></tr><tr><td>聚乙烯亚胺（PEI） </td><td> Sigma-Aldrich公司</td><td> 408727 </td></tr><tr><td>限制性内切酶</td><td> NEB </td><td>各个</td></tr><tr><td> T4 DNA连接酶</td><td> NEB </td><td> M0202T </td></tr><tr><td>凝胶提取试剂盒</td><td> Qiagen公司</td><td> 28704 </td></tr><tr><td> phusion二聚合酶套件</td><td> finnzymes（NEB） </td><td>的F-540S </td></tr><tr><td> HotStar高保真聚合酶试剂盒</td><td> Qiagen公司</td><td> 202602 </td></tr><tr><td>二甲基亚砜</td><td> finnzymes（NEB） </td><td>的F-540S（套件的一部分） </td></tr><tr><td> EDTA（25毫米） </td><td> Invitrogen公司</td><td> 18068-015（套件的一部分） </td></tr><tr><td>三</td><td>罗斯</td><td> 4855.2 </td></tr><tr><td>氨苄西林钠盐</td><td>罗斯</td><td> K029.2 </td></tr><tr><td> dNTPs浓度（10毫米，100微升） </td><td> Invitrogen公司</td><td> 18427013 </td></tr><tr><td>碘克沙醇（OptiPrep） </td><td>轴盾</td><td> 1114739 </td></tr><tr><td> Phenolred </td><td>默克公司</td><td> 107241 </td></tr><tr><td>巨型质粒准备套件</td><td> Qiagen公司</td><td> 12181 </td></tr><tr><td>超速离心机</td><td>贝克曼 - 库尔特</td><td>舰L90K </td></tr><tr><td>快速密封离心管</td><td>贝克曼 - 库尔特</td><td> 342414 </td></tr><tr><td>电单位</td><td> BIO-RAD </td><td> GenePulserXcell </td></tr><tr><td>热循环仪</td><td> Eppendorf公司</td><td> VAPO保护</td></tr><tr><td>加热块</td><td>博日</td><td> MB-102 </td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> IX81 </td></tr><tr><td>流式细胞仪</td><td>贝克曼 - 库尔特</td><td> cytomics FC500的MLP </td></tr><tr><td> MegaX DH10B T1R细胞</td><td> Invitrogen公司</td><td> C640003 </td></tr><tr><td> benzonase </td><td>默克公司</td><td> 101695 </td></tr><tr><td>腺病毒5 </td><td> ATCC </td><td> VR-5 </td></tr><tr><td>载体pBluescript二KS（+）质粒</td><td> Stratagene公司</td><td> 212207 </td></tr><tr><td> cap5F（PAC我在网站黄色，cap5的特定序列大胆）： GACTCTTAATTAACAGGT ATGTCTTTTGTTGATCACCCTCC </td><td> IDTDNA </td><td>自定义底漆</td></tr><tr><td> cap5R（ASC绿色，在的大胆cap5特定序列）在我的网站： GTGAGGGCGCGCC TTAAAGGGGTCGGGTAAGGTATC </td><td> IDTDNA </td><td>自定义底漆</td></tr></tbody></table>

engineering and evolution of synthetic adeno-associated virus (aav) gene therapy vectors via dna family shuffling

腺相关病毒（AAV），向量表示由于1有益特性的独特组合，一些用于治疗人类的基因转移的最有力的和有前途的车辆。包括底层野生型病毒和非常先进的方法生产高滴度，高纯度和临床级2重组载体的apathogenicity。另一项特别的优势超过其他病毒的腺相关病毒系统是一个自然发生的血清型，其中不同的基本属性，都可以很容易地设计载体，使用一个共同的协议1,2财富的可用性。此外，包括我们自己的团体最近已制定战略，利用这些天然病毒作为模板创建的合成相结合的多输入血清型的资产，或提高单个孤立的属性载体。各自的技术，以实现这些目标ARé任的DNA家族改组3， 即由他们根据部分同源性（通常&gt; 80％，大多数腺相关病毒血清型），或肽显示4,5， 即通常7个氨基酸插入到重新组装各种AAV衣壳基因 ​​的碎片理想肽介导的重新定位到所需的细胞类型的病毒衣壳的暴露循环。为了获得最大的成功，这两种方法都适用于高吞吐量的时尚据此协议缩放产生大约100万不同的衣壳变种库。每个克隆，然后组成一个独特的组合（DNA改组方法）众多家长病毒或病毒在同一骨干（肽显示方法）包含一个独特的肽。在随后的最后一步是迭代选择这样的靶细胞库，以丰富个人履行或理想的选择过程中的所有要求衣壳。后者最好梳INES积极的压力，如在某些细胞类型，利益与负选择，例如消除所有与抗AAV抗体反应衣壳增长。这种组合增加合成衣壳生存选择，在给定的应用程序可能会发现在任何自然发生的腺相关病毒分离的方式相匹配的需求的机会。在这里，我们专注于DNA家族改组方法的理论和实验两种技术的挑战。我们将介绍和演示洗牌腺相关病毒库的生成和选择（ 图1）所有必要的步骤，然后讨论存在的隐患和协议的重要方面，是一个需要知道为了成功与腺相关病毒的分子进化。

Watch this Scientific Journal Video about Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling at JoVE.com

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

我们展示了基本的技术，分子工程和发展合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组。此外，我们在文化或靶细胞在小鼠的增强属性的个体嵌合衣壳的选择和分析，并提供一般的指引，有代表性的例子。

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Research

JoVE Journal

Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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 ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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

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 ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

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Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

传播<em&gt; Homalodisca coagulata病毒01</em&gt;通过<em&gt; Homalodisca vitripennis</em&gt;细胞培养

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...