Homologous recombination involving small single-stranded oligonucleotides in human cells

Gene modification by homologous recombination is one of the techniques that may eventually be used in gene replacement therapy. We tested whether small, synthetic single-stranded oligodeoxynucleotides are capable of participating in homologous recombination in human cells. A plasmid carrying a mutant neomycin phosphotransferase (neo) gene was cotransfected with a 40-nucleotide single-stranded oligomer that contained the wild-type neo gene sequence into human cells. Cells expressing neo were selected in the antibiotic G418. These cells contained wild-type molecules, which resulted from recombination between the two molecules. The results indicate that this approach may be useful in correcting or introducing single point mutations into the genomes of mammalian cells.