Production of viral vectors using recombinase-mediated cassette exchange
Production of viral vectors using recombinase-mediated cassette exchange
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DOI：
10.1093/nar/gni074
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Kanegae, Y
中科院分区：
文献类型：
--
作者：
Nakano, M;Odaka, K;Kanegae, Y
DNA病毒通常用作外源基因表达的载体，但通常必须处理来自克隆或真实病毒基因组的大DNA区域以产生病毒载体。在这里，我们提出了一种独特的系统，通过使用重组酶介导的盒式交换（RMCE）反应，用表达 Cre 的 293 细胞中复制病毒基因组中的替换基因直接替换小转染质粒中的目的基因，从而生成腺病毒载体。结合与目的基因连接的病毒顺式作用包装信号的正选择，目的载体在感染三个和四个周期后分别富集到97.5%和99.8%。我们的结果还表明，突变体 loxP V（以前称为 loxP 2272）（RMCE 反应中使用的 Cre 变体靶标）可用作野生型 loxP 的不相容突变体。该方法不仅可用于同时生成大量腺病毒载体，还可用于生成其他DNA病毒载体，包括辅助依赖性腺病毒载体。
DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.