Meanwhile, the results of the competition analyses suggested that loxP insertion, not only at 191 nt but also at 143 nt, possibly affected the efficiency of virus packaging. Among the six pairs of loxP-containing viruses, we chose 15L and 19L for the competition assay because the difference in the ratio of the viral titers for these viruses was the smallest (Table 2); thus, this difference probably had a minimal effect on the competition analysis. Furthermore, the differences

in the viral growth between 15L, 19L or ΔL and the competitor may reflect a difference in packaging efficiency. Although the titer of the competitor after the seventh passage was higher than not only that of 19L, but also that of 15L, this difference was not observed in the competition analysis. For the competitor virus, the ratio of the titer in the seventh stock versus Erismodegib chemical structure that

in the conventional stock (6.7 in Table 1) was slightly higher than that for 15L, 19L and ΔL, thereby suggesting that the replication efficiency of the competitor virus might be effective. However, while the titer of 15L alone was identical to that of ΔL (both 3.2 in Table 1) and the ratio of ΔL + competitor did not change during the seventh passage, the decrease in the ratio of the 15L + competitor in the competition analysis was remarkable (Figs. 3a,b). mTOR inhibitor Therefore, because these decreases did not depend on the replication efficiency, these results suggested that the insertion of loxP upstream

of the cis-acting packaging domain influenced the packaging step. One second report has claimed that a virus lacking the region from 53 nt to 322 nt at the left-end of the virus genome showed a packaging efficiency that was nearly comparable to that of the wild type (19), suggesting that these insertions may not influence the packaging efficiency. Although we examined the effect of loxP insertion only at 143 nt or 191 nt, because the loxP sequence is a palindrome structure, the insertion of such a sequence might actively hamper the binding of some factor, thereby disturbing the packaging to the same extent. This negative effect of loxP insertion is probably a useful characteristic for a helper virus in HD-AdV construction. During the construction of HD-AdV, the incomplete excision of the packaging domain of a helper virus in Cre-expressing 293 cells remains a very important problem: approximately 5% of helper virus persists in crude HD-AdV stocks (33, 34). Such incomplete excision might result from the toxicity of highly expressed Cre in 293 cells (35–38) or from a shut-off mechanism for Cre expression during vector replication (33). FLP and FLPe, which is a thermo-stabilized FLP, have also been applied for this purpose, and their excision efficiencies were reportedly similar to or a little more than that of Cre (16, 17).