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.

We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence called loxP. Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters. The resulting 293Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive "molecular switch." High efficiency recombination was observed for Ad viral DNA containing loxP sites. The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination between loxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined by loxP sites in viral genomes.