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 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.
Fujita
A
A
Department of Molecular Biology, University of Tokyo, Japan.
Sakagami
K
K
Kanegae
Y
Y
Saito
I
I
Kobayashi
I
I
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics*; D018274:Electroporation; D004926:Escherichia coli; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D027383:Papillomaviridae / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D016662:Virus Integration
D017931:DNA Primers; D004279:DNA, Viral; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase