Fig. 1.

PhiBT1 integration system. (A) Schematic representation of site-specific phage DNA integration into host genome. The phage integrase recognizes the attB and attP sites in the bacterial and phage genomes, respectively, as substrates and catalyzes the recombination reaction between them, resulting in the integration of phage DNA into the host genome. This is a unidirectional reaction because the products of recombination, attL and attR, are not recognized by the integrase. In nature, the integrated phage DNA can only be rescued from the bacterial genome by action of excisionase, which is a distinct phage-encoded enzyme that recognizes attL and attR sequences as substrates for recombination. (B) Schematic maps of plasmids. Plac, lacZ promoter; chl, chloramphenicol resistant gene; int, phiBT1 integrase gene; kan, kanamycin resistant gene; CMV, CMV promoter; NLS, SV40 nuclear localization signal; CAG, CAG promoter; IRES, the internal ribosome entry site from the encephalomyocarditis virus (ECMV); SEAP, reporter gene secreted alkaline phosphatase; mPAH, mouse PAH gene cDNA. (C) SEAP expression in transfected mouse 3T3 cells after serial passaging. Mouse 3T3 cells were cotransfected with the integrase-expressing plasmids (pCMV-BTInt or pCMV-BTIntNLS) and reporter plasmids without (pCZiS) or with (pCZiS-B) an attB sequence at the ratio of 20:1. SEAP concentrations in conditioned media of all groups rose to similar levels after transfection, which returned to background in the attB-negative group after serial passaging. Those in the attB-positive groups, however, remained stable at a reduced level after multiple cell passages (P < 0.05 for normal integrase group, and P < 0.01 for NLS-containing integrase group). Additionally, the presence of SV40NLS sequence in the integrase enzyme resulted in a 4-fold higher level of SEAP expression in the transfected cells after serial passaging (P < 0.05).