MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering |
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Authors: | Kaja Kostyrko Samuel Neuenschwander Thomas Junier Alexandre Regamey Christian Iseli Emanuel Schmid‐Siegert Sandra Bosshard Stefano Majocchi Valérie Le Fourn Pierre‐Alain Girod Ioannis Xenarios Nicolas Mermod |
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Affiliation: | 1. Department of Fundamental Microbiology, Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL‐EPFL, Lausanne, Switzerland;2. Swiss Institute of Bioinformatics, Lausanne, Switzerland;3. Selexis SA, Geneva, Switzerland;4. +41‐21‐693‐61‐51+41‐21‐693‐76‐10 |
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Abstract: | Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. |
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Keywords: | DNA recombination microhomology‐mediated end‐joining Chinese hamster ovary cells recombinant protein expression immunoglobulin production |
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