Abstract
Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.
Full Text
The Full Text of this article is available as a PDF (533.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Chinnadurai G., Chinnadurai S., Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol. 1979 Nov;32(2):623–628. doi: 10.1128/jvi.32.2.623-628.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engelhardt J. F., Ye X., Doranz B., Wilson J. M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6196–6200. doi: 10.1073/pnas.91.13.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ginsberg H. S., Lundholm-Beauchamp U., Horswood R. L., Pernis B., Wold W. S., Chanock R. M., Prince G. A. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci U S A. 1989 May;86(10):3823–3827. doi: 10.1073/pnas.86.10.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
- Gräble M., Hearing P. Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant. J Virol. 1990 May;64(5):2047–2056. doi: 10.1128/jvi.64.5.2047-2056.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gräble M., Hearing P. cis and trans requirements for the selective packaging of adenovirus type 5 DNA. J Virol. 1992 Feb;66(2):723–731. doi: 10.1128/jvi.66.2.723-731.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hearing P., Shenk T. Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit. Mol Cell Biol. 1985 Nov;5(11):3214–3221. doi: 10.1128/mcb.5.11.3214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
- Jones N., Shenk T. Isolation of deletion and substitution mutants of adenovirus type 5. Cell. 1978 Jan;13(1):181–188. doi: 10.1016/0092-8674(78)90148-4. [DOI] [PubMed] [Google Scholar]
- Kellum R., Schedl P. A position-effect assay for boundaries of higher order chromosomal domains. Cell. 1991 Mar 8;64(5):941–950. doi: 10.1016/0092-8674(91)90318-s. [DOI] [PubMed] [Google Scholar]
- McGrory W. J., Bautista D. S., Graham F. L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. doi: 10.1016/0042-6822(88)90302-9. [DOI] [PubMed] [Google Scholar]
- Munz P. L., Young C. S. End-joining of DNA fragments in adenovirus transfection of human cells. Virology. 1991 Jul;183(1):160–169. doi: 10.1016/0042-6822(91)90129-y. [DOI] [PubMed] [Google Scholar]
- Stillman B. W. The replication of adenovirus DNA with purified proteins. Cell. 1983 Nov;35(1):7–9. doi: 10.1016/0092-8674(83)90201-5. [DOI] [PubMed] [Google Scholar]
- Zhang W. W., Fang X., Branch C. D., Mazur W., French B. A., Roth J. A. Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques. 1993 Nov;15(5):868–872. [PubMed] [Google Scholar]