Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction.Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.Complete viral genomes of large DNA viruses (e.g. herpesviruses) can be incorporated as bacterial artificial chromosomes (BACs). One method is the use of replicons based on the bacterial F plasmid [1]. Since the initial report that described the BAC cloning of a murine cytomegalovirus genome [2], several additional herpesvirus genomes have been cloned as BACs [3-14], including herpes simplex virus 1 (HSV-1) and herpes simplex 2 (HSV-2) [15-18].BAC technology created new possibilities for the targeted mutagenesis of the herpesvirus genome (for recent reviews, see [19,20]). Since the viral genome can be carried as an episome in bacterial cells, prokaryotic genetic techniques can be employed for manipulation of the viral genome without the need for typical selective mechanisms required for mutagenesis and selection in eukaryotic cells. This allows for the generation of viral mutants that would be difficult to obtain otherwise, either because of the lack of a phenotypic effect due to mutation or because the mutation confers a disadvantage for selection and replication in eukaryotic cells. Viral particles are o