Figure 2. Schematic diagrams and Southern blot analyses of the three HSV-1 mutants. Southern blot analysis of CEAICP4 and d 120 DNA digested with Nru I using an ICP6 probe revealed the presence of the transgene insertion into the ICP6 gene locus. The 900-bp fragment observed in d 120 is expected in the absence of homologous recombination, whereas the 4.7-kb fragment observed in CEAICP4 results from integration of transgene sequences into the ICP6 gene locus. The presence of the CEA promoter construct as well as of the upstream CEA flanking sequence was confirmed by hybridization of Bam HI-digested DNA with probes to PCEA3 and CEALF. CEAICP4 contains the expected 0.6-kb fragment in the case of the PCEA3 probe and the expected 0.7-kb and 1.5-kb fragments in the case of the CEALF probe, none of which are present in d 120 DNA. Lastly, the presence of the correct ICP4 fragments in CEAICP4 was confirmed by hybridization of Bam HI-digested DNA with the 3.2-kb Hinc II fragment of ICP4 as probe. CEAICP4 maintains the ICP4 gene deletions of the d 120 backbone (1.1-kb and 3.2-kb fragments) and in addition contains the ICP4 transgene insertion into the ICP6 locus, resulting in two new fragments (1.7-kb and 2.3-kb) hybridizing to an ICP4 probe. Southern blot analysis of R3616 and CEAγ34.5 DNA digested with Nru I using an Nru I fragment of ICP6 as a probe reveals the expected 888-bp band in R3616 and the expected 6.7-kb band in CEAγ34.5 that results from transgene insertion into the ICP6 locus. Nru I-digested R3616 and CEAγ34.5 DNA examined with a γ134.5 probe reveals no hybridization in R3616 as expected and a 6.7-kb band in CEAγ34.5 as a result of homologous recombination of γ134.5 into the ICP6 gene locus. Southern blot analysis of F strain and DF3γ34.5 DNA digested with Pst I using a thymidine kinase (TK) probe reveals a 0.8-kb band of the native TK gene locus in F strain but a 4.4-kb band in DF3γ34.5 as a result of homologous recombination, with insertion of γ134.5 sequences into the TK gene locus. When hybridized with a DF3 promoter sequence probe, only the expected 4.4-kb band is observed in DF3γ34.5 DNA, and as expected no band is observed in F strain DNA.