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PubMed · 7666520

Gene targeting with a replication-defective adenovirus vector.

A Fujita·K Sakagami·Y Kanegae·I Saito·I Kobayashi
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Abstract

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 gene 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 viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants 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 of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy.

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AdenoviridaeAnimalsBase SequenceCell LineCloning, MolecularDNADNA PrimersDNA, ViralDefective VirusesElectroporationEscherichia coliGenetic VectorsKanamycin KinaseMiceModels, StructuralMolecular Sequence DataNucleic Acid ConformationPapillomaviridaePhosphotransferases (Alcohol Group Acceptor)PlasmidsRecombination, GeneticSequence Homology, Nucleic AcidTransfectionVirus IntegrationComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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A Fujita, K Sakagami, Y Kanegae, I Saito, I Kobayashi. 1995. Gene targeting with a replication-defective adenovirus vector.. https://doi.org/10.1128/jvi.69.10.6180-6190.1995

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