Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
J Genet Genomics. 2012 Jun 20;39(6):275-80. doi: 10.1016/j.jgg.2012.04.004. Epub 2012 May 9.
The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.
大鼠是许多生物医学研究和药物开发领域首选的动物模型。由于缺乏有效的基因靶向工具，大鼠的基因操作一直落后于小鼠。之前，我们通过大鼠胚胎干细胞（ES）细胞中的常规同源重组产生了敲除大鼠。在这里，我们表明通过转录激活因子样效应物核酸酶（TALEN）介导的 DNA 双链断裂，可以快速实现大鼠 ES 细胞中的高效基因靶向。使用 Golden Gate 克隆技术，我们在 5 天内构建了一对用于感兴趣基因的 TALEN 靶向载体。基因转染后，无需药物选择即可通过 PCR 分离、筛选和确认靶向大鼠 ES 细胞集落。我们的结果表明，TALEN 介导的基因靶向是一种高效、快速周转时间的建立基因修饰大鼠 ES 细胞系的优越方法。
