Figure 1.

(A) Schematic representation of vector structures. All rep cassettes were constructed and inserted in the helper-dependent plasmid pRP1030 as outlined in Materials and Methods. Mutation of Rep52/40 ATG to GGA, G → A mutation of splice donor site, and deletion of first Rep78 ATG (respectively nucleotides 993–996, 1,907, and 321–323 of AAV-2) were reported on the vector diagrams. pRP1030 (shown in linear form) contains sequences corresponding to left ends (base pairs 3–466) and to the right end (base pairs 35,464–35,924) of Ad5 genome including ITRs and packaging signal. All other Ad5 genomic sequences were deleted and substituted with a mouse cytomegalovirus immediate early promoter (MCMV)-lacz cassette that corresponds to nucleotides 467–4,920 of pRP1030, a 22,425-bp BglII fragment of λ DNA (nucleotides 5,128–27,138). A bacterial plasmid (pMX2) is present between nucleotides 27,138 and 29,373. (B) Serial amplification of Hd-Rep vectors. pRP1030 was amplified as positive control. All plasmids were converted to linear molecules and packaged into infectious virions after transfection of 293CRE4 cells infected with AdLC8 helper virus. Amplification was measured by 293 infection with aliquots of 293CRE lysate and β-galactosidase staining.