Genetic Manipulation of Poxviruses Using Bacterial Artificial Chromosome Recombineering

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.