Zhou et al. constructed new helper viruses carrying loxP at 143 nt in AflIII or at 192 nt in BsrGI and at 358 nt (26). Maeda et al. also reported helper

viruses carrying the upstream loxP at 143 nt or at 192 nt and the downstream loxP at 358 nt or at 454 nt, and a helper virus lacking the region from 192 nt to 358 nt generated by the Cre/loxP deletion was capable of growing in 293 cells to some extent, indicating that viral packaging occurred using only the A-repeats of AVI and AVII present between 358 nt and 454 nt (24). Thus, the 454-nt check details position appears to be better than the 358-nt position for the downstream insertion of loxP in the helper virus because all seven A-repeats present between 194 nt and 380 nt (19) are removed by Cre/loxP deletion. Regarding the upstream insertion site of loxP, the above-mentioned authors reported that neither the loxP site at 143 nt nor at 192 nt influenced viral growth and that the obtained titer of

the virus carrying loxP at 143 nt appeared lower than that of a virus carrying loxP at 192 nt (24, 26). However, both groups examined only one pair of viruses. Our results described here were different from theirs. The results of six pairs of viruses that were tested for titration showed that the titers of 15L AdV for high titer were not lower than that of 19L viruses and even much higher for low titer viruses (Table click here 2). The difference became remarkable for the high passage stock (Table 1, column 7). As for comparison of 15L Digestive enzyme and 19L with ΔL in a competition assay, a very sensitive method recognized in the virological field, clearly showed that the loxP insertion of both 15L and 19L did slightly influence the viral growth and the packaging that depends on the growth. This difference may have arisen because the assays used in the present report were more sensitive than those used previously and possibly because the method used in Zhou et al. (26) is different from ours: they used a method of one-step growth curve only in a particular passage of MOI 0.5, while we

examined time courses of the passages. Interestingly, the difference in titers between 15L and 19L was sometimes remarkable when the titers of these viruses were very low (see Table 2, the bottom two pairs), possibly because multiple rounds of infection to surrounding cells are necessary. A high titer helper virus would be advantageous for the generation of HD-AdV. Zhou et al. (26) reported that helper viruses possessing an intact E3 region showed approximately 5–10-fold more yield of HD-AdV probably because of more efficient complementation. Therefore, a helper virus containing loxP at 143 nt is possibly more useful than that at 191 nt. In fact, we obtained results that more HD-AdV was produced when 15L-type helper viruses were used (data not shown).