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First published online November 17, 2005
Stem Cells Vol. 24 No. 3 March 2006, pp. 615 -623
doi:10.1634/stemcells.2005-0226; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS

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

David M. Suter, Laetitia Cartier, Esther Bettiol, Diderik Tirefort, Marisa E. Jaconi, Michel Dubois-Dauphin, Karl-Heinz Krause

Biology of Aging Laboratory, Department of Rehabilitation and Geriatrics, University of Geneva Medical School, Geneva, Switzerland

Key Words. Lentivector • Embryonic stem cells • Recombinational cloning • Neuronal differentiation • Promoter/reporter construct • Antibiotic selection • 2K7

Correspondence: David Suter, M.D., Laboratory of Aging Biology, 2 chemin du Petit-Bel-Air, 1225 Chêne-Bourg, Switzerland. Telephone: +41-223-055-453; Fax: +41-223-055-455; e-mail: david.suter{at}hcuge.ch

Received May 18, 2005; accepted for publication September 6, 2005.
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.




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E. Aberdam, E. Barak, M. Rouleau, S. de LaForest, S. Berrih-Aknin, D. M. Suter, K.-H. Krause, M. Amit, J. Itskovitz-Eldor, and D. Aberdam
A Pure Population of Ectodermal Cells Derived from Human Embryonic Stem Cells
Stem Cells, February 1, 2008; 26(2): 440 - 444.
[Abstract] [Full Text] [PDF]


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