A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase.
A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase.
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DOI：
10.1016/j.jviromet.2013.10.023
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发表时间：
2014-03
影响因子：
3.1
通讯作者：
Taniguchi K
中科院分区：
文献类型：
--
作者：
Komoto S;Kawagishi T;Kobayashi T;Ikizler M;Iskarpatyoti J;Dermody TS;Taniguchi K
哺乳动物正呼肠孤病毒（呼肠孤病毒）已成为研究病毒复制和发病机制的非常有用的模型。基于质粒的反向遗传学系统的开发代表了呼肠孤病毒研究的重大突破。目前用于呼肠孤病毒的反向遗传学系统依赖于在用呼肠孤病毒基因片段cDNA质粒转染的细胞内T7 RNA聚合酶的表达。在这些系统中，通过使用表达T7 RNA聚合酶的重组牛痘病毒或组成型表达T7 RNA聚合酶的细胞系来提供T7 RNA聚合酶。在这里，我们描述了一种替代的基于质粒的救援系统驱动的质粒编码的T7 RNA聚合酶，这可以增加这种反向遗传学系统的灵活性。尽管这种方法需要转染另外的质粒，但当A549、BHK-21或L929细胞与呼肠孤病毒10-质粒组和编码T7 RNA聚合酶的质粒一起共转染时，实现了病毒回收。从理论上讲，该系统提供了在任何细胞系中产生呼肠孤病毒的可能性，包括适于增殖用于临床用途的病毒载体的细胞系。因此，这种方法将增加用于呼肠孤病毒生物学基础研究的反向遗传学的灵活性，并促进用于临床应用的呼肠孤病毒的开发。
Mammalian orthoreoviruses (reoviruses) have served as highly useful models for studies of virus replication and pathogenesis. The development of a plasmid-based reverse genetics system represented a major breakthrough in reovirus research. The current reverse genetics systems for reoviruses rely on the expression of T7 RNA polymerase within cells transfected with reovirus gene-segment cDNA plasmids. In these systems, the T7 RNA polymerase is provided by using a recombinant vaccinia virus expressing T7 RNA polymerase or a cell line constitutively expressing T7 RNA polymerase. Here, we describe an alternative plasmid-based rescue system driven by a plasmid-encoded T7 RNA polymerase, which could increase the flexibility of such reverse genetics systems. Although this approach requires transfection of an additional plasmid, virus recovery was achieved when A549, BHK-21, or L929 cells were co-transfected with a reovirus 10-plasmid set together with a plasmid encoding T7 RNA polymerase. Theoretically, this system offers the possibility to generate reoviruses in any cell line, including those amenable to propagation of viral vectors for clinical use. Thus, this approach will increase the flexibility of reverse genetics for basic studies of reovirus biology and foster development of reoviruses for clinical applications.
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影响因子：
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