...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Nucleic Acids Research >Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.
【24h】

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

机译：通过稳定的标签交换基因靶向将精确的改变高效导入小鼠基因组：对ES细胞中基因靶向的影响。

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

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. A modified approach is described, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kbof homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for a beta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. These results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.
机译：用于引入精确基因组修饰的标签和交换基因靶向方法的效率受到高水平的非同源重组体的损害，所述非同源重组体由于标签基因表达的丧失而常常在选择中幸免，通常是由于标签基因的物理损失。描述了一种被称为稳定标签交换的改进方法，该方法结合了额外的正选择（稳定性）盒以规避该限制。通过4.9 kb同源DNA分开的HPRT（标签）和neo（稳定性）盒被有效地引入ES细胞的LIF基因座。在交换步骤靶向中，标签盒取代了β-半乳糖苷酶基因。标记和交换方法与稳定的标记交换方法的直接比较表明，各自的靶向效率分别为21％和88％。稳定的标签交换靶向效率的提高是由于消除了> 75％的背景品系，这些背景品由于标签基因的物理缺失而在标签和交流选择中幸存下来。这些归因于将标记的等位基因还原为野生型，因此是标记和交换靶向背景的主要贡献者。这些结果通过证明扩展的非同源性的多个区域的单步整合的原理，并为将精确的改变重复引入哺乳动物基因组提供了有效的系统，扩展了基因靶向的应用。

著录项

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