Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters.
Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.
Mittal
S K
SK
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Bett
A J
AJ
Prevec
L
L
Graham
F L
FL
D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology; D004261:DNA Replication; D015870:Gene Expression; D005798:Genes, Bacterial; D017344:Genes, Insect; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis*; D008322:Mammals; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D008156:Luciferases; D001616:beta-Galactosidase