Gene Targeting Mediated By Triple Helix Forming Oligonuc

由三螺旋形成寡核苷酸介导的基因打靶

• 批准号: 6668135
• 负责人: Michael M SEIDMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6668135
• 关键词: gene mutation; gene targeting; method development; nucleic acid sequence; oligonucleotides; tissue /cell culture; triple helix; gene mutation; gene targeting; method development; nucleic acid sequence; oligonucleotides; tissue /cell culture; triple helix

We are developing a gene targeting program based on the capacity of oligonucleotides to form stable triple helix complexes with specific sequence in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. Triple helices have been known for over 40 years and have been the subject of many studies in vitro. However there is direct evidence that the protein:nucleic acid structure of mammalian chromosomes would preclude access to triplex forming oligos (TFO). We prepared a TFO linked to a photoactivatable DNA mutagen directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. We have prepared TFOs with novel sugar modifications that show enhanced targeting activity. Treatment of S phase cells with these TFOs results in 30% of targeted crosslinking and 5-10% mutation frequencies, while both crosslinking and mutagenesis are much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. Furthermore the frequency of mutagenesis is sufficiently high to allow identification of colonies with sequence changes in simple screens of a few clones.

我们正在基于寡核苷酸在双链DNA中具有特定序列的稳定三重螺旋络合物的能力开发基因靶向程序。这种方法有望成为一种简单有效的技术，用于将DNA反应性化合物传递到活细胞中染色体DNA的特定位点。应用包括基因敲除，定向基因转化和重组，以及基因治疗。三重螺旋已闻名已有40多年，并且是许多体外研究的主题。然而，有直接的证据表明，蛋白质：哺乳动物染色体的核酸结构将排除进入三寡寡核酸（TFO）的访问。我们准备了与经常用作突变报告基因（HPRT）中序列的光活化DNA诱变剂相关的TFO。我们将其引入哺乳动物细胞，并在诱变剂光活化后，分离出具有突变的细胞菌落。序列分析表明，突变位于基因内的目标序列。我们已经用新的糖修饰制备了TFO，这些TFO显示出增强的靶向活性。用这些TFO对S相细胞的处理导致30％的靶向交联和5-10％的突变频率，而交联和诱变均在静态细胞中都低得多。这些结果表明，哺乳动物细胞中染色体靶位点的可及性受细胞的生物学调节。此外，诱变的频率足够高，可以识别几个克隆简单筛选中序列变化的菌落。