Nucleic Acids Research Advance Access originally published online on August 5, 2008
Nucleic Acids Research 2008 36(17):e111; doi:10.1093/nar/gkn508
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Nucleic Acids Research, 2008, Vol. 36, No. 17 e111
© 2008 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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Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
*To whom correspondence should be addressed. Tel: +49 30 94062596; Fax: +49 30 94062551; Email: mgossen{at}mdc-berlin.de
Received April 30, 2008. Revised July 10, 2008. Accepted July 24, 2008.
Nonuniform, mosaic expression patterns of transgenes are often linked to transcriptional silencing, triggered by epigenetic modifications of the exogenous DNA. Such phenotypes are common phenomena in genetically engineered cells and organisms. They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation. Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns. Standard antibiotic selection was directly compared to cell sorting for the establishment of stable cells. Only the latter procedure could warrant a high degree of uniformity and stability in gene expression. Marker genes useful for the essential cell sorting step encode mostly fluorescent proteins. However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest.
Present address: Weimin L. Kaufman, Center for Pediatric Biomedical Research, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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