03 was used BI-6727 (Fig. 3b, 1–8, 13–20). Because 15L and 19L have the same structure as ΔL except for the loxP insertion at 141 nt

and 191 nt, respectively, these negative effects were probably due to the loxP insertion upstream of the packaging domain. To visualize each marker gene expression, HeLa cells were infected with the fifth stocks of a mixture of 15L + competitor (corresponding to Figs. 3a,b, lanes 3). When an initial competitor ratio of 1:0.3 was used, the β-gal expression of the 15L virus mostly disappeared and only a small number of cells were stained (Fig. 3c, upper left panel; also see Fig. 3a lane 3). Meanwhile, the GFP expression produced by the competitor virus was amply detected in the majority of cells at various intensities (lower left panel; see Fig. 3a, lane 15). When an initial competitor ratio of 1:0.03 was used, the β-gal expression of 15L persisted in most of the cells and significant, but weak, GFP expression was detected (Fig. 3c, right panels; also see Fig. 3b, lanes 3 and 15). These result were consistent with the virus genome copy numbers in the 293 cells from the fourth passage (Fig. 2b, lane 3) and showed that the loxP insertion in both the 15L

and 19L viruses had a deleterious effect on the competition experiments. We showed that the titers of 15L and 19L containing Target Selective Inhibitor Library loxP upstream of the cis-acting packaging domain AI were similar to ΔL, though 19L possessing a loxP insertion at 191 nt sometimes produced a slightly lower titer than that of ΔL and 15L. Because the virus titer probably reflects

the final number of infectious viral particles in the stock, namely, the end-point of the amount of functional viral particles in the valance between viral growth and inactivation, this result suggested that the loxP insertion at 191 nt may influence the viral growth. Meanwhile, in the competition experiments that are thought, at least partly, to reflect the efficiencies of the packaging of the viral genome and the transmission of the virus, both the 15L and 19L viruses carrying loxP at 143 nt and 191 nt were gradually out-competed with every passage and were completely replaced by the competitor virus that did not contain loxP after only four passages. These results clearly showed that these the loxP insertion in the upstream region outside the packaging domain caused a negative effect on viral packaging. We also constructed AdV called 15F and 19F, which contains FRT, the target sequence of FLP, instead of loxP. The titer of 15F was 5.6-fold higher than that of 19F (data not shown), indicating that the insertion of FRT caused a similar effect. Therefore, it was suggested that at least these recombinase targets influenced the viral growth and packaging, though we have no data to answer whether the effect is specific for loxP and FRT or a sequence other than the recombinase targets. Viruses containing loxP insertions upstream and downstream of the packaging domain have already been reported as helper viruses.