Transient dominant selection for the modification and generation of recombinant infectious bronchitis coronaviruses.
Transient dominant selection for the modification and generation of recombinant infectious bronchitis coronaviruses.
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DOI：
10.1007/978-1-4939-2438-7_12
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Britton P
中科院分区：
文献类型：
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作者：
Keep SM;Bickerton E;Britton P
我们已经开发了一种针对禽冠状病毒传染性支气管炎病毒（IBV）的反向遗传学系统，其中对应于IBV基因组的全长cDNA在T7启动子序列的控制下插入到牛痘病毒基因组中。作为全长IBV cDNA的载体的牛痘病毒具有的优点是，可以使用同源重组将修饰引入IBV cDNA中，同源重组是一种经常用于从牛痘病毒基因组插入和缺失序列的方法。在这里，我们描述了使用瞬时显性选择作为一种方法引入修改到IBV的cDNA，这已成功地用于特定核苷酸的取代，删除基因组区域，和交换完整的基因。感染性重组IBV是在将含有修饰的IBV cDNA的牛痘病毒DNA转染到用表达T7 DNA依赖性RNA聚合酶的重组鸡痘病毒感染的细胞中后原位产生的。
We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA; this has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.