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Adenovirus Vectors, Site Specific Recombinases and Molecular Switches used to Regulate Gene Expression

Control of gene expression from Ad vectors

There are numerous systems for regulating gene expression, and many have been used in Ad vectors. However, most (if not all) of these are leaky, and allow at least a low level of expression from cassettes in the “off” mode. As a consequence certain cytotoxic genes cannot be rescued into Ad vectors. Furthermore, even gene products that are not necessarily cytotoxic can be inhibitory when over expressed and may result in the cassette being difficult or impossible to rescue into an Ad vector. Molecular “switches” based on excision of a DNA segment inserted between a promoter and a cDNA can be engineered to be extremely “tight”, as the “stuffer” DNA can include transcriptional and translational stop signals that prevent gene expression. When the intervening stuffer is excised expression can be turned on to very high levels if a strong promoter such as the HCMV or MCMV promoter is used.


Expression of the reporter gene can be switched on by infecting a host cell that expresses the Cre recombinase1 or by coinfection of cells with a second Ad vector that expresses Cre2,3. Examples of vectors expressing Cre recombinase are AdCre1 (Figure 2A) and AdCreM1 (Figure 2B) which express Cre under the control of the HCMV IE gene promoter and the MCMV IE gene promoter respectively.

First generation (FG) Ad vectors expressing Cre recombinase have all the advantages of Ad vectors, namely growth to high titres, ease of purification, efficient gene transfer resulting in high level gene expression in virtually any mammalian cell type and efficient gene delivery to a variety of different tissues when injected into animals. The transient nature of gene expression from FG Ad vectors is also advantageous for most applications using Cre.



Figure 4. In vivo gene expression from a molecular switch regulated by Cre recombinase expressed by AdCre

Because Ad vectors can be used for efficient gene delivery into many tissues in most animals the use of AdCre in vivo makes possible an entirely new approach to using transgenic animals in research: “somagenics” in which delivery and expression of recombinases can be used to remodel the genome in somatic tissues of transgenics carrying floxed DNA (Figure 4). The possible applications are too numerous to list but for a few examples of studies that have used AdCre in transgenic animals see References 4 and 5.

Cre expression using the AdCreM2 vector is driven by the murine Cytomegalovirus immediate early gene promoter (MCMV) instead of the HCMV promoter used in AdCre1. Dr. Graham has shown that the MCMV promoter results in higher expression levels in almost all cells compared to the HCMV promoter which is relatively weak in rodent cells especially (Addison, C. L., Hitt, M., Kunsken, D. and Graham, F. L. Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression in adenoviral vectors. J. Gen. Virol. 78:1653-1661, 1997.) . Consequently, he uses the vector with the MCMV promoter (AdCreM2). AdCreM2 is not yet described in the literature

Kit G is covered by US patent nos. 6,974,694 and 6,120,764

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1 Chen, L., Anton, M. and Graham, F. L. Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre. Somat. Cell and Molec. Genet. 22: 477-488, 1996.)

2 Anton, M. and Graham, F. L. Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression. J. Virol. 69:4600-4606, 1995

3 Wang, P., Anton, M., Graham, F. L. and Bacchetti, S. High frequency recombination between LoxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase. Somatic Cell and Molec. Genet. 21: 429-441, 1995.

4 Wang, Y., Krushel, L. A. and Edelman, G. M. Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. Proc. Natl. Acad. Sci US 93: 3932-3936, 1996;

5 Rohlmann, A., Gotthardt, M., Willnow, T. E., Hammer, R. E. and Herz, J. Sustained somatic gene inactivation by viral transfer of Cre recombinase. Nature Biotech. 14: 1562-1565, 1996