Control of Gene Expression from Ad Vectors Using Molecular Switches Triggered by Recombinase
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 from Ad vectors 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 unless the stuffer DNA segment is excised. When the intervening stuffer is excised expression can be turned on to very high levels if a strong promoter such as the HCMV (human cytomegalovirus) immediate early gene promoter or MCMV (murine cytomegalovirus) promoter is used. An example is the use of coinfection of cells with two Ad vectors, one expressing Cre and a second containing a cassette comprising the LacZ gene whose expression is dependent on Cre recombinase. Cre activity excises the spacer between the promoter and the LacZ coding sequences resulting in expression of beta-galactosidase (Figure 1 and 2).
Figure 2. Expression of the reporter gene can be switched on by infecting a host cell that expresses the Cre recombinase (1,2) or, as below by coinfection of cells with a second Ad vector that expresses Cre (3).
Examples of vectors expressing Cre recombinase are AdCre1 (Figure 3A) and AdCreM1 (Figure 3B) which express Cre under the control of the HCMV IE gene promoter and the MCMV IE gene promoter respectively.
An important use of Ad vectors expressing Cre recombinase takes advantage of the many useful properties of Ad vectors, namely growth to high titres and 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. An additional property of gene delivery by “first generation” (FG) Ad vectors, not usually considered an advantage, is the transient nature of gene expression which is actually advantageous in certain circumstances.
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 transgenic animals 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 3 -5 below.
For more detailed information contact DMKS Research (DMKS_Ltd@shaw.ca).
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
References
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