Infectious delivery of Alphaherpesvirus bacterial artificial chromosomes

Bacterial artifi cial chromosomes (BACs) can accommodate and stably propagate the genomes of large DNA viruses in E. coli . As DNA virus genomes are often per se infectious upon transfection into mammalian cells, their cloning in BACs and easy modifi cation by homologous recombination in bacteria has become an important strategy to investigate the functions of individual virus genes. This chapter describes a strategy to clone the genomes of viruses of the Alphaherpesvirinae subfamily within the family of the Herpesviridae , which is a group of large DNA viruses that can establish both lytic and latent infections in most animal species including humans. The cloning strategy includes the following steps: (1) Construction of a transfer plasmid that contains the BAC backbone with selection and screening markers, and targeting sequences which support homologous recombination between the transfer plasmid and the alphaherpesvirus genome. (2) Introduction of the transfer plasmid sequences into the alphaherpesvirus genome via homologous recombination in mammalian cells. (3) Isolation of recombinant virus genomes containing the BAC backbone sequences from infected mammalian cells and electroporation into E. coli . (4) Preparation of infectious BAC DNA from bacterial cultures and transfection into mammalian cells. (5) Isolation and characterization of progeny virus