Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly
Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly
复制标题
DOI：
10.1002/0471142727.mb1626s113
复制
发表时间：
2016-01
影响因子：
--
通讯作者：
Yong Yang;Yudan Chi;Xinying Tang;H. Ertl;Dongming Zhou
中科院分区：
文献类型：
--
作者：
Yong Yang;Yudan Chi;Xinying Tang;H. Ertl;Dongming Zhou
腺病毒载体作为基因转移载体和疫苗载体在基础研究和临床应用中取得了可喜的成果。然而，构建腺病毒载体和操纵腺病毒（Ad）基因组的最常见程序是复杂且劳动密集型的。一个简单而详细的协议，快速，高效，和模块化的黑猩猩Ad血清型68（AdC68）作为一个分子克隆通过等温组装，定向组装多个DNA片段在一个单一的等温反应，没有限制性内切酶或连接酶，提出。任何血清型的腺病毒基因组序列已知，都可以用这种方法构建成分子克隆。重组腺病毒载体可以通过一步等温组装在<3天内产生，并且重组Ad可以在8天内被拯救。该方案对于Ad基因组的操作是实用的，因为整个Ad基因组可以被分成模块质粒内的特定片段。John Wiley & Sons，Inc.
Adenoviral vectors have yielded promising results as carriers for gene transfer and vaccines in basic research and clinical applications. However, most common procedures to construct adenoviral vectors and manipulate adenovirus (Ad) genomes are complex and labor‐intensive. An easy and detailed protocol for the rapid, efficient, and modular generation of chimpanzee Ad serotype 68 (AdC68) as a molecular clone via isothermal assembly, which directionally assembles multiple DNA fragments in a single isothermal reaction without restriction enzymes or ligases, is presented. Any serotype of adenovirus with the sequence of genome known can be constructed as a molecular clone by this method. Recombinant adenoviral vectors can be created via one‐step isothermal assembly in <3 days, and recombinant Ads can be rescued within 8 days. This protocol is practical for manipulations of Ad genomes, because an entire Ad genome can be divided into specific fragments within modular plasmids. © 2016 by John Wiley & Sons, Inc.