Fig. 4.

Generation of ΔB-S rEBV. Shown is the recombineering strategy used to generate rEBV mutant ΔB-S, which is deleted for the entire BHLF1 ORF and 5′ promoter region up to the right (3′) boundary of the deletion in the EBV genomes within the Sal BL cell line (23); the deletion also removed the leftmost lytic-cycle origin of DNA replication (oriLyt). Left, agarose gel analysis of BamHI-digested and corresponding Southern blot of the parental Ak-BAC-GFP (lane 1), intermediate BAC clone containing the targeting cassette FRT-rpsL-tet-FRT in place of deleted EBV DNA (lane 2), and the final BAC clone of ΔB-S (lane 3) after Flp-mediated removal of the targeting cassette, resulting in a 3.3-kbp deletion and insertion of one Flp target sequence (FRT). Right, configuration of the domain targeted within the EBV genome. Shown are the BamHI restriction sites (vertical arrows) defining the BamHI-Y (1.8-kbp) and BamHI-H (6.0-kbp) restriction fragments of the wt EBV genome (top) and within the intermediate (middle) and final (bottom) mutated EBV BAC clones. In addition to BamHI-H and BamHI-Y, diagnostic 2.8- and 2.9-kbp BamHI restriction fragments expected to hybridize to the probe are indicated below each DNA construct (right) and by white dots on the ethidium bromide-stained agarose gel (left). Open arrows depict the ORFs present in the targeted domain. The DNA diagram is not to scale.