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First published online November 17, 2005
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2005-0226v1
24/3/615    most recent
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Submitted on May 18, 2005
Accepted on September 6, 2005

Original Article

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

David M. Suter 1*, Laetitia Cartier 1, Esther Bettiol 1, Diderik Tirefort 1, Marisa E. Jaconi 1, Michel Dubois-Dauphin 1, Karl-Heinz Krause 1

1 Biology of Aging Laboratory, Dept. of Rehabilitation and Geriatrics, University of Geneva Medical School, Geneva, Switzerland

* To whom correspondence should be addressed. E-mail: David.Suter{at}hcuge.ch.


  Abstract

Generation of stable transgenic embryonic stem (ES) cell lines by classical 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 to construct lentivectors and to generate 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 cPPT and WPRE elements as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system: i) is functional in mouse and human embryonic stem cells; ii) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters iii) allows to obtain ES cells expressing two constructs through selection with different antibiotics. The technology described in this article should become a useful tool in stem cell research.

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




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