Figure 2

Construction of the recombinant adenovirus AdNY58. The bacteriophage lambda LIA7 was recovered from the MutaMouse by in vitro packaging. An SmaI-SacI fragment of LIA7 within its lacZ gene was inserted into pIK153. The Tyr105Stop mutation (Figure 3) was introduced into the resulting plasmid (pIK153LZS.6) using site-directed mutagenesis by PCR as follows. The PCR products generated with the primer pair LZT-U (5'-CGAAGAGGCCCGCAC-3') and LZT-MA (5'-TAATGGGCTAGGTTACGTTGGTGTAG-3'), and the primer pair LZT-MS (5'-TAACCTAGCCCATTACGGTCAATCC-3') and LZT-D (5'-GGCAACATGGAAATCGC-3') were mixed and used as templates for the second PCR with the primer pair LTZ-U and LZT-D. Replacement of an FspI-AatII fragment of pIK153LZS.6 by the FspI-AatII fragment of the resulting PCR product resulted in pIK153 T10.1. A BamHI-SmaI fragment covering the lacZ gene of LIA7 was inserted into the BamHI site of pIK153 (resulting in pNY19). pNY21 was made by replacing the smaller SmaI-SacI fragment of pNY19 with the homologous SmaI-SacI fragment of pIK153T10.1, which carries the mutant sequence. An XbaI-BglII fragment of pNY21 was used to replace the smaller XbaI-BamHI fragment of pHM5 (resulting in pNY58). pAdNY58 was made by replacement of the smaller I-CeuI-PI-SceI fragment of pAdHM4 with an I-CeuI-PI-SceI fragment of pNY58. The longer PacI fragment of pAdNY58 was transfected into 293 cells. The recombinant adenovirus AdNY58 was prepared and purified from the cell culture.