Fig. 1. Schematic representation of recombinant HSVLatEnk vector and its molecular characterization. Thymidine kinase gene (UL23) in KOS-derived Tk+HSVLatβ-gal DNA [bearing β-galactosidase reporter gene downstream of the Lat-LTR promoter inserted into the gC locus (Antunes-Bras et al., 1998)] was disrupted by homologous recombination with p23d plasmid DNA containing NsiI toSacI deleted HSV TK gene to generate TK-negative HSVLatβ-gal vector (HSVLatβ-gal). HSVLatEnk recombinant was then created as described previously (Antunes-Bras et al., 1998), by inserting Lat-LTR-pEnk transcriptional unit into the gC gene of HSVLatβ-gal by homologous recombination. Purified HSVLatEnk DNA was analyzed by PCR, and subsequent PCR products were separated on ethidium bromide-stained 1.2% agarose gel for the presence of both the TK-deleted gene and the Enk transgene. Positions of respective primers used are indicated on the diagram. Lanes 1 and6 show the Lambda DNA/HindIII-EcoRI and 100 bp molecular weight standards (given in kilobases), respectively. Lane 2, Approximately 1.8 kb PCR product generated by amplification, using primers A/B, of wild-type HSV DNA; lane 3, ∼1.3 kb PCR product generated using the same primers A/B on HSVLatEnk DNA.Lanes 4 and 5 show the PCR amplification products obtained with the set of primers 1/3 and 2/3 on HSVLatEnk DNA, respectively. HSVLatEnk DNA was further analyzed by Southern hybridization. Ten micrograms of HSVLatβ-gal DNA (lane 7), 10 μg of HSVLatEnk DNA (lane 8), and 0.1 μg of pLatEnk DNA (lane 9) were digested withBamHI, applied on 0.8% agarose gel, electrotransferred on nylon membrane, and hybridized with a [32P]-labeled cDNA probe corresponding to the rat pEnk coding region. Whereas no hybridization signal was apparent on HSVLatβ-gal (lane 7), positively labeled DNA fragments of ∼1.35 kb were generated on HSVLatEnk DNA (lane 8) and pLatEnk DNA (lane 9). This size corresponds to the expected size of DNA fragment resulting from theBamHI digestion of both HSVLatEnk and pLatEnk DNA.