Turner P C, Moyer R W
Department of Immunology and Medical Microbiology, University of Florida, Gainesville 32610-0266.
Biotechniques. 1992 Nov;13(5):764-71.
A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.
描述了一种改变痘苗病毒（VV）特定基因的通用方法。我们通过基因失活来演示和评估该程序，使用显性选择标记与重组聚合酶链反应（PCR）相结合。基于靶基因序列的引物能够扩增侧翼臂，并随后将其连接到赋予对霉酚酸抗性的gpt盒上。线性PCR构建体被转染到感染野生型痘苗病毒的细胞中。然后通过在MPA存在下生长来选择通过同源重组将gpt插入靶基因的突变病毒。该技术应用于痘苗病毒胸苷激酶基因，并与通过克隆构建含gpt质粒的传统方法进行比较。发现PCR方案非常高效，理论上可用于将任何外源DNA元件插入任何非必需靶基因，只要有该基因的部分或完整序列信息即可。该程序有可能用于多种基因修饰，包括将外源基因插入痘病毒和其他DNA病毒。像细菌和酵母这样可以用线性DNA转化的微生物基因组，也是这种方法操作的候选对象。
