title: MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.
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.
authors: Department of Fundamental Microbiology, Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland.
authors: Kaja
authors: K
authors: Kostyrko
authors: Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors: Samuel
authors: S
authors: Neuenschwander
authors: Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors: Thomas
authors: T
authors: Junier
authors: Selexis SA, Geneva, Switzerland.
authors: Alexandre
authors: A
authors: Regamey
authors: Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors: Christian
authors: C
authors: Iseli
authors: Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors: Emanuel
authors: E
authors: Schmid-Siegert
authors: Department of Fundamental Microbiology, Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland.
authors: Sandra
authors: S
authors: Bosshard
authors: Department of Fundamental Microbiology, Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland.
authors: Stefano
authors: S
authors: Majocchi
authors: Selexis SA, Geneva, Switzerland.
authors: Valérie
authors: V
authors: Le Fourn
authors: Selexis SA, Geneva, Switzerland.
authors: Pierre-Alain
authors: PA
authors: Girod
authors: Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors: Ioannis
authors: I
authors: Xenarios
authors: Department of Fundamental Microbiology, Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland.
authors: Nicolas
authors: N
authors: Mermod
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002843:Chromatin / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D055785:Gene Knockdown Techniques; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D045170:Matrix Attachment Regions / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
chemical_list: D000906:Antibodies; D002843:Chromatin; D011994:Recombinant Proteins
keywords: Chinese hamster ovary cells; DNA recombination; immunoglobulin production; microhomology-mediated end-joining; recombinant protein expression