MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

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Ressource 1Télécharger: Kostyrko_et_al-2016-Biotechnology_and_Bioengineering.pdf (1674.77 [Ko])
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ID Serval
serval:BIB_322831BD76E3
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.
Périodique
Biotechnology and Bioengineering
Auteur(s)
Kostyrko K., Neuenschwander S., Junier T., Regamey A., Iseli C., Schmid-Siegert E., Bosshard S., Majocchi S., Le Fourn V., Girod P.A., Xenarios I., Mermod N.
ISSN
1097-0290 (Electronic)
ISSN-L
0006-3592
Statut éditorial
Publié
Date de publication
2017
Peer-reviewed
Oui
Volume
114
Numéro
2
Pages
384-396
Langue
anglais
Résumé
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.

Mots-clé
DNA recombination, microhomology-mediated end-joining, Chinese hamster ovary cells, recombinant protein expression, immunoglobulin production
Pubmed
Web of science
Open Access
Oui
Création de la notice
30/11/2016 11:18
Dernière modification de la notice
20/08/2019 13:17
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