Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation

• Published in: Nucleic acids research Vol. 36; no. 17; p. e111
• Main Authors: Kaufman, Weimin L, Kocman, Ibrahim, Agrawal, Vishal, Rahn, Hans-Peter, Besser, Daniel, Gossen, Manfred
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.10.2008; Oxford Publishing Limited (England)
• Subjects: Animals; Anti-Bacterial Agents - pharmacology; Cell Line; Cell Separation; CHO Cells; Clone Cells; Cricetinae; Cricetulus; Flow Cytometry; Gene Expression; HeLa Cells; Humans; Methods Online; Transfection; Transgenes
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkn508

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.