| Summary: | Therapeutic proteins require proper folding and post-translational modifications (PTMs) to be effective and biologically active. Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. Ubiquitous Chromatin Opening Elements (UCOEs) are DNA elements naturally found upstream of specific housekeeping genes, which are proposed to maintain open chromatin structure, supporting stable and high-level transgene expression by prevention of transgene silencing. In this study we have examined the interaction between UCOE and DHFR-linked amplification in relation to cell expression stability. CHO-DG44 cell lines were engineered to express erythropoietin (EPO) or a green fluorescent protein (GFP) from constructs with or without the inclusion of a UCOE. Cell lines were amplified in the presence of 250 nM metotrexate (MTX) and were then grown continuously for over 70 days in the presence and absence of MTX. Growth characteristics, protein expression, plasmid copy numbers, mRNA expression, karyotype and recombinant gene localisation (by fluorescent in situ hybridisation - FISH) were assessed for cells at stages throughout the period of long-term culture. In summary the inclusion of UCOE elements generated cells that; • achieved higher cell densities and exhibited increased production of recombinant mRNA/cell and protein yield • allowed isolation of greater numbers of high producing clones • resulted in greater mRNA recovery/recombinant gene copy• retained stable mRNA and protein expression after amplification provided MTX was present (but not in the absence of MTX when instability was observed) • exhibited a more consistent karyotype and no abnormal chromosomal rearrangements. It was concluded that the inclusion of UCOEs within expression constructs offer significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR.
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