GENE TARGETING WITH A REPLICATION-DEFECTIVE ADENOVIRUS VECTOR

Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gen e inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their vi ability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosph ate procedure. The structure of the recombinants was analyzed in detai l after recovery in an Escherichia coli strain, All of the recombinant s examined had experienced a precise correction of the mutant neo gene . Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end o f the donor-target homology. The vector adenovirus DNA was inserted in to some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a conco mitant insertion of a 10-bp-long filler sequence in one of the recombi nants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the a pplicability of adenovirus-mediated gene delivery in gene targeting an d gene therapy.