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Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals f lanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an Ϸ10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of Ϸ10 10 transducing virions͞ml, with <0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.

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No abstract

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3 (PI-3) kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3 kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3 kinase were highly apoptotic, suggesting that recruitment of PI-3 kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3 kinase binding site was noted in tumors derived from the strains expressing the PI-3 kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3 kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3 kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.Mammary epithelial cell-specific expression of the polyomavirus (PyV) middle T (MT) oncogene in transgenic mice results in the induction of multifocal metastatic mammary tumors involving 100% of the transgene carriers (19). The potent oncogenic properties of the PyV MT are due to its ability to associate with and activate a number of cellular signaling proteins. One class of cellular enzymes activated by PyV MT consists of members of the Src family tyrosine kinases (c-Src and c-Yes) (8,12,27,30). Expression of PyV MT in mammary glands of Src-deficient mice rarely results in the induction of mammary tumors (20), suggesting that activation of Src by PyV MT is required for mammary tumorigenesis. Whereas activation of c-Src is required for the rapid induction of mammary tumors, this event is not sufficient, because expression of a constitutively active version of Src in the mammary ...