Suter David M, Cartier Laetitia, Bettiol Esther, Tirefort Diderik, Jaconi Marisa E, Dubois-Dauphin Michel, Krause Karl-Heinz
Biology of Aging Laboratory, Department of Rehabilitation and Geriatrics, University of Geneva Medical School, Switzerland.
Stem Cells. 2006 Mar;24(3):615-23. doi: 10.1634/stemcells.2005-0226. Epub 2005 Nov 17.
Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.
通过经典转染生成稳定的转基因胚胎干细胞（ES）系仍然是一项艰巨的任务，需要耗时的克隆选择，并且受到克隆假象和基因沉默的阻碍。在此，我们描述了一种新型系统，该系统允许构建慢病毒载体，并在非常短的时间内生成转基因表达率> 99%的稳定ES细胞系。通过模块化重组克隆系统可快速插入启动子和感兴趣的基因。载体包含来自HIV-1元件的中央多聚嘌呤序列和土拨鼠肝炎病毒转录后调控元件以及抗生素抗性，以实现最佳和均匀的转基因表达。我们表明该系统：1）在小鼠和人类ES细胞中具有功能；2）允许生成在遍在或组织特异性启动子控制下表达感兴趣基因的ES细胞；3）允许通过用不同抗生素进行选择获得表达两种构建体的ES细胞。本文所述技术应成为干细胞研究中的一种有用工具。
