title: Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.
abstract: The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.
authors.affiliation: The University of Manchester, Faculty of Life Sciences, Michael Smith Building, Manchester, United Kingdom. zeynep.betts@kocaeli.edu.tr.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Betts
authors.affiliation: The University of Manchester, Faculty of Life Sciences, Michael Smith Building, Manchester, United Kingdom.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Dickson
mesh_terms: D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000235:genetics*; D033481:Chromosomes, Mammalian / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005784:Gene Amplification; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D002843:Chromatin; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008727:Methotrexate
keywords: Chinese hamster ovary (CHO) cells; Chromosome structure; Methotrexate (MTX) amplification; Recombinant protein production; Ubiquitous Chromatin Opening Elements (UCOE)