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

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 tra...

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Autores principales: Kaufman, Weimin L., Kocman, Ibrahim, Agrawal, Vishal, Rahn, Hans-Peter, Besser, Daniel, Gossen, Manfred
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553579/
https://www.ncbi.nlm.nih.gov/pubmed/18682524
http://dx.doi.org/10.1093/nar/gkn508
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author Kaufman, Weimin L.
Kocman, Ibrahim
Agrawal, Vishal
Rahn, Hans-Peter
Besser, Daniel
Gossen, Manfred
author_facet Kaufman, Weimin L.
Kocman, Ibrahim
Agrawal, Vishal
Rahn, Hans-Peter
Besser, Daniel
Gossen, Manfred
author_sort Kaufman, Weimin L.
collection PubMed
description 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.
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spelling pubmed-25535792008-10-01 Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation Kaufman, Weimin L. Kocman, Ibrahim Agrawal, Vishal Rahn, Hans-Peter Besser, Daniel Gossen, Manfred Nucleic Acids Res Methods Online 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. Oxford University Press 2008-10 2008-08-05 /pmc/articles/PMC2553579/ /pubmed/18682524 http://dx.doi.org/10.1093/nar/gkn508 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ 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.
spellingShingle Methods Online
Kaufman, Weimin L.
Kocman, Ibrahim
Agrawal, Vishal
Rahn, Hans-Peter
Besser, Daniel
Gossen, Manfred
Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title_full Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title_fullStr Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title_full_unstemmed Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title_short Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
title_sort homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553579/
https://www.ncbi.nlm.nih.gov/pubmed/18682524
http://dx.doi.org/10.1093/nar/gkn508
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