...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells
【24h】

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

机译：CAG重复序列中锌指定向的双链断裂促进人细胞中的重复不稳定

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAGCTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the Fokl DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.
机译：扩大的三联体重复序列已被确定为越来越多的神经系统疾病和骨骼疾病的遗传基础。为了检查双链断裂修复对哺乳动物系统中CAGCTG重复序列不稳定性的贡献，我们开发了识别和切割CAG重复序列的锌指核酸酶（ZFN）。工程ZFN使用串联的锌指阵列与Fokl DNA切割域融合，以位点特异性方式指导双链断裂（DSB）。我们首先确定ZFN切割CAG在体外重复。然后，使用我们先前描述的组织培养测定法来鉴定CAG重复序列不稳定性的修饰子，我们发现ZFN表达载体的转染在人和啮齿动物细胞系中诱导CAG重复序列的变化最多增加了15倍，并且更长重复序列比短重复序列对分裂更敏感。治疗后出现的单个菌落的分析显示，一系列事件与ZFN诱导的DSB一致，并以重复收缩为主。我们还发现，与ZFN结合表达RAD51的显性阴性形式，可显着降低核酸酶的作用，这表明DSB诱导的重复不稳定性部分是通过同源性定向修复介导的。这些研究将ZFN鉴定为表征DSB对细胞中CAG重复序列作用的有用试剂。

著录项

  • 来源
  • 作者

    David Mittelman; Christopher Moye; Jason Morton; Kristen Sykoudis; Yunfu Lin; Dana Carroll; John H. Wilson;

  • 作者单位

    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030;

    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030;

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112;

    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030;

    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030;

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112;

    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    DNA repair; gene targeting; triplet repeat instability; zinc finger nucleases;

    机译：DNA修复;基因靶向三联体重复不稳定性;锌指核酸酶;

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号