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: 7132325
• 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/7132325
• 关键词: gene mutation; gene targeting; method development; nucleic acid sequence; oligonucleotides; tissue /cell culture; triple helix

We are developing a gene targeting program based on oligonucleotides that form stable triple helix complexes with specific sequences 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. We have prepared TFOs containing novel sugar analogues and have identified a modification format that supports efficient targeting of specific chrosomal sequences in living mamalian cells. In our developmental work we prepared TFOs, linked to a photoactive 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. Treatment of S phase cells with these TFOs results in 30% of targeted crosslinking and 5-10% mutation frequencies. 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. This strategy has been extended to other genes for which genetic analyses of targeting are not possible. For example, we have recently shown, using a biochemical assay, that a site in the human beta globin gene can be targeted at high efficiency. We also find that the targeting oligonucleotides can be used to direct homologous recombination. Cells treated with the TFO and a donor DNA with homology to the region around the target sequence show knock-in of the donor DNA at frequencies 3-4 orders of magnitude above that seen with cells treated with donor alone. We have studied the genetic requirements for repair and mutagenesis of the targeted crosslinks. We find a requirement for the ERCC1/XPF complex for targeted base substitutions. In contrast the appearance of deletion mutations is independent of all nucleotide excision repair, double strand break repair, and recombinational repair genes.

我们正在开发基于基于寡核苷酸的基因靶向程序，该寡核苷酸与双链DNA中的特定序列形成稳定的三重螺旋络合物。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。我们已经准备了含有新糖类似物的TFO，并确定了一种修饰格式，该格式支持在活马乳细胞中有效靶向特定的玻璃序列。在我们的发展工作中，我们准备了与光活性DNA诱变剂相关的TFO，该TFO针对经常用作突变报告基因（HPRT）中的序列。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。用这些TFO处理S相细胞会导致30％的靶向交联和5-10％的突变频率。在静态细胞中，交联和诱变都低得多。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。该策略已扩展到其他基因，无法实现靶向的遗传分析。例如，我们最近使用生化测定法表明，人β球蛋白基因中的位点可以针对高效率。我们还发现，靶向寡核苷酸可用于引导同源重组。用TFO处理的细胞和与目标序列周围区域同源的供体DNA处理的细胞显示，供体DNA在频率3-4个数量级的频率高于单独用供体处理的细胞中看到的频率3-4个数量级。我们研究了靶向交联的修复和诱变的遗传要求。我们发现针对目标基本取代的ERCC1/XPF复合物的要求。相反，缺失突变的出现与所有核苷酸切除修复，双链断裂修复和重组修复基因无关。