Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors

被引:86
作者
Suter, David M. [1 ]
Cartier, Laetitia [1 ]
Bettiol, Esther [1 ]
Tirefort, Diderik [1 ]
Jaconi, Marisa E. [1 ]
Dubois-Dauphin, Michel [1 ]
Krause, Karl-Heinz [1 ]
机构
[1] Univ Geneva, Sch Med, Dept Rehabil & Genet, Lab Aging Biol, CH-1225 Chene Bourg, Switzerland
关键词
lentivector; embryonic stem cells; recombinational cloning; neuronal differentiation promoter/reporter construct; antibiotic selection; 2K7;
D O I
10.1634/stemcells.2005-0226
中图分类号
Q813 [细胞工程];
学科分类号
摘要
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.
引用
收藏
页码:615 / 623
页数:9
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