Cottingham Matthew G
The Jenner Institute, University of Oxford, Oxford, UK.
Methods Mol Biol. 2012;890:37-57. doi: 10.1007/978-1-61779-876-4_3.
Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥ 500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories.
痘病毒基因操作的传统方法依赖于病毒感染细胞中的低频自然重组。尽管这些强大的系统代表了当前痘病毒知识和应用的技术基础，但它们需要长（≥500 bp）的侧翼序列用于同源重组、一种有效的病毒筛选方法以及繁琐且耗时的蚀斑纯化。随着阿尔班·多米和伯纳德·莫斯发明了长期以来一直寻求的获得痘苗病毒BAC克隆（VAC-BAC）的方法，21世纪初见证了细菌人工染色体（BAC）技术在痘病毒中的应用，作为其基因操作的替代方法。BAC系统的关键优势在于使用噬菌体λ Red重组（重组工程）进行基因操作的简便性和通用性，该方法仅需要约50 bp的同源臂，可通过PCR轻松创建，并且允许在无需进行瞬时显性选择的情况下产生无任何标记基因的无缝突变。另一方面，也存在一些缺点，包括显著的设置时间、克隆基因组被细菌插入序列污染的风险以及从衍生病毒中去除BAC盒的棘手问题。必须仔细权衡这些因素，以决定使用BAC对于特定应用是否有利，这使得痘病毒BAC系统在大多数实验室中可能是对传统方法的补充，而非替代。
