Rapid Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors

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...

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Bibliographic Details
Published in:Stem cells (Dayton, Ohio) Ohio), 2006-03, Vol.24 (3), p.615-623
Main Authors: Suter, David M., Cartier, Laetitia, Bettiol, Esther, Tirefort, Diderik, Jaconi, Marisa E., Dubois‐Dauphin, Michel, Krause, Karl‐Heinz
Format: Article
Language:English
Subjects:
2K7
Animals
Antibiotic selection
Cell Line
Embryo, Mammalian - cytology
Embryo, Mammalian - physiology
Embryonic stem cells
Gene Expression - genetics
Genetic Vectors
Human immunodeficiency virus 1
Humans
Lentivector
Lentivirus
Mice
Neuronal differentiation
Promoter/reporter construct
Recombination, Genetic - genetics
Recombinational cloning
Regulatory Elements, Transcriptional - genetics
Stem Cells - cytology
Stem Cells - physiology
Transduction, Genetic - methods
Transgenes - genetics
Woodchuck hepatitis virus
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Summary: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.
ISSN:1066-5099
1549-4918
DOI:10.1634/stemcells.2005-0226