Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
1] Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany [2].
Nat Commun. 2014;5:3045. doi: 10.1038/ncomms4045.
Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.
利用同源重组将转基因引入小鼠模型是理解人类疾病基础生物学和病理学的关键。在这里，我们研究了人工转录激活因子样效应物核酸酶（TALENs）——一种可在特定基因组位置诱导 DNA 双链断裂的强大工具——是否可以与靶向载体结合，以诱导胚胎干细胞和受精卵中的同源重组，从而引入转基因。我们描述了一种使用 TALENs 生成条件性小鼠模型的方法，该方法在特殊 AT 富含序列结合蛋白-1 的基因组位点诱导双链断裂，同时使用一个大的 14.4 kb 靶向模板载体。我们报告了该等位基因成功的种系传递，并在 Cre 重组酶存在的情况下证明了其在原代细胞中的重组。这些结果表明，TALEN 辅助诱导的 DNA 双链断裂可以促进同源重组复杂的靶向构建直接在卵母细胞中。
