Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides
Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides
复制标题
DOI：
10.1073/pnas.1218721110
复制
发表时间：
2013-03-05
影响因子：
11.1
通讯作者：
Kuehn, Ralf
中科院分区：
文献类型：
--
作者：
Wefers, Benedikt;Meyer, Melanie;Kuehn, Ralf
对遗传疾病机制的研究主要依赖于源自工程胚胎干细胞（ES）的靶向小鼠突变体。然而，突变ES细胞的建立既费力又耗时，限制了对高通量基因组分析所发现的日益增多的人类疾病突变的研究。在此，我们提出一种先进的方法，通过将转录激活因子样效应物核酸酶（TALENs）和合成寡脱氧核苷酸显微注射到单细胞胚胎中来制备小鼠疾病模型。在胚胎注射后的2天内，我们在小GTP酶RAB38中创造并纠正了错义突变；RAB38是细胞内囊泡运输的调节因子，也是赫曼斯基 - 普德拉克综合征的表型模型。由于不需要ES细胞培养和靶向载体，该技术能够实现即时的生殖系修饰，使杂合突变体在18周内即可获得。通过TALENs和寡脱氧核苷酸直接诱变的关键特征，即省力和快速，促进了未来用于研究人类疾病机制和干预措施的体内模型的产生。
The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.