Although this region acts partly as an E1A enhancer in wild-type Ad5, the enhancer function is not necessary because a sufficient amount of E1A proteins are supplied in 293 cells. The loxP-insertion site at 191 nt of SgrAI in AdLC8cluc, which is the most popular helper Deforolimus virus, is extremely close to the cis-acting packaging domain AI described above. The virus

titers of helper viruses containing loxP at 143 nt in the AflIII cleavage site or at 192 nt in the BsrGI site have been studied (24, 26) (Fig. 1a). These previous reports suggested that the titer of the virus carrying loxP at 192 nt was slightly higher than, or not different to, that of the virus carrying loxP at 143 nt. However, both groups examined

only one pair of the viruses and so far no detailed examinations have since been performed. In this report, we constructed six pairs of AdV containing upstream loxP at 143 nt or 191 nt; we then compared the resulting virus titers and examined the influence of the loxP insertion FK228 cost upstream of the packaging domain AI. We observed that the viral titers of the AdV containing loxP at 143 nt was not lower and sometimes much higher than those of the AdV containing loxP at 191 nt. In a competition analysis, where two different viral genomes compete to be packaged into a viral shell, the insertion of loxP at both 143 nt and 191 nt reduced the packaging efficiency, compared with that of the competing AdV which did not contain loxP. These results suggested that the upstream insertion of loxP influences viral packaging. The human embryonic kidney cell line, 293, was cultured in DMEM supplemented Adenosine with 10% FCS. HeLa cells, derived from human cervical cancer, were also cultured in 10% FCS-DMEM. After infection with AdV, the cells were maintained in 5% FCS-DMEM. To examine the influence of loxP insertion near the packaging

domain AI, we constructed a new AdV, which has one loxP at 143 or 191 nt, a LacZ-expression unit, and another loxP at 466 nt, in this order (Fig. 1a). The left-end fragment of the Ad5 genome including a loxP at the AflIII site (143 nt), at which the loxP is located at approximately 150 nt after Klenow polymerase treatment, or at the SgrAI site (191 nt) was introduced into the cassette cosmid pAxcw (27); the former and latter positions were named here as 15L and 19L, respectively. The terms 15L and 19L also refer to the names of the viruses containing loxP at these sites. The resultant cosmid was termed pAx15Lcw or pAx19Lcw, respectively. The expression unit, expressing the LacZ gene under the control of the human polypeptide EF1α promoter (28), and the second loxP in this order were inserted at the SwaI site (464 nt; 454 nt in the original Ad5 genome), which is located downstream of the repeat AIIV in pAx15Lcw or pAx19Lcw; the resulting cosmid was named pAxLEFZ15L or pAxLEFZ19L, respectively.