Zhang Xu, Ferreira Irene R S, Schnorrer Frank
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Methods. 2014 Aug 15;69(1):32-7. doi: 10.1016/j.ymeth.2014.03.020. Epub 2014 Mar 26.
Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2 months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.
从保种中心可获得数千种点突变、缺失或转座子插入。然而，迄今为止，以特定方式修饰特定基因座仍然困难。一种潜在的解决方案是应用转录激活样效应物核酸酶（TALENs），其已成功用于在各种模式生物中使基因突变。TALENs通过将TALE蛋白与核酸内切酶FokI融合构建而成，产生人工的、序列特异性核酸内切酶。它们诱导双链断裂，双链断裂可通过易出错的非同源末端连接（NHEJ）或同源定向修复（HDR）进行修复。我们开发了一种基于TALEN的简单方案，可在约2个月内使果蝇中任何感兴趣的基因突变。我们将编码靶向同一基因的两对TALEN的mRNA注射到胚胎中，利用T7核酸内切酶I对混合的F1代果蝇进行筛选以鉴定突变，并在F3代中产生稳定的突变品系。我们通过使CG11617突变来说明我们策略的有效性，CG11617是果蝇中一个功能未知的先前未表征的假定转录因子。这表明TALENs是使果蝇中任何感兴趣的基因突变的可靠且高效的策略。
