Nucleic Acids Research Advance Access originally published online on September 22, 2006
Nucleic Acids Research 2006 34(18):e120; doi:10.1093/nar/gkl674
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Nucleic Acids Research, 2006, Vol. 34, No. 18 e120
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Transduction of human embryonic stem cells by ecotropic retroviral vectors
Institute of Reconstructive Neurobiology, Life and Brain Center University of Bonn and Hertie Foundation, Bonn, Germany 1 Department of Obstetrics and Gynecology, Rambam Medical Center Haifa, Israel
*To whom correspondence should be addressed at Institute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. Tel: +49 228 6885 500; Fax: +49 228 6885 501; Email: brustle{at}uni-bonn.de
Received August 11, 2006. Accepted August 31, 2006.
The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.
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