title: Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation.
abstract: 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.
authors.affiliation: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
authors.forename: Weimin L
authors.initials: WL
authors.lastname: Kaufman
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Kocman
authors.forename: Vishal
authors.initials: V
authors.lastname: Agrawal
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Rahn
authors.forename: Daniel
authors.initials: D
authors.lastname: Besser
authors.forename: Manfred
authors.initials: M
authors.lastname: Gossen
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D014162:Transfection; D019076:Transgenes
chemical_list: D000900:Anti-Bacterial Agents