Triplex-Stimulated Intermolecular Recombination at a Single-Copy Genomic Target

详细信息    查看全文

• 作者：Melissa P. Knauert ; Jennifer M. Kalish ; Denise C. Hegan and Peter M. Glazer
• 关键词：triplex-forming oligonucleotide ; homologous recombination ; gene correction ; phosphoramidate ; firefly luciferase ; oligonucleotide
• 刊名：Molecular Therapy
• 出版年：2006
• 出版时间：September 2006
• 年：2006
• 卷：14
• 期：3
• 页码：392-400
• 全文大小：397 K

文摘

Gene targeting via homologous recombination offers a potential strategy for therapeutic correction of mutations in disease-related human genes. However, there is a need to improve the efficiency of site-specific recombination by transfected donor DNAs. Oligonucleotide-mediated triple helix formation has been shown to constitute a DNA lesion sufficient to provoke DNA repair and thereby stimulate recombination. However, the ability of triplex-forming oligonucleotides (TFOs) to induce recombination between a target locus and a donor DNA has so far been demonstrated only with multicopy episomal targets in mammalian cells. Using cell lines containing the firefly luciferase reporter gene, we have now established the ability of TFOs to induce gene correction by exogenous donor DNAs at a single-copy chromosomal locus. We find that cotransfection of TFOs and short, single-stranded DNA donor molecules into mammalian cells yields gene correction in a dose-dependent manner at frequencies up to 0.1 % , which is five- to ninefold above background. We demonstrate both oligonucleotide-specific and target site-specific effects. We also find that recombination can be induced by both parallel and antiparallel triple helix formation. These results provide further support for the development of TFOs as reagents to stimulate site-specific correction of defective human genes.