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Abstract

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

“…In contrast, a decrease in the cellspecific productivity was observed in two of the non-UCOE cell lines. These results are consistent with previous studies, where inclusion of UCOE in the vector construct resulted in higher levels of recombinant protein production [19,21,27,28,30] and improved the transgene expression in hematopoietic stem cells [25,26]. Otte et al compared the effect of different DNA elements, including UCOEs, S/MARs, cHS4 insulator and anti-repressor elements, on protein expression levels [37].…”

“…When MTX selection was required, MTX was added to a final concentration of 250 nM. 19 Hypoxanthine and Thymidine (HT) solution (Gibco) was added to the growth medium for DHFR-deficient non-transfected parental CHO-DG44 cells. Cells were cultured routinely in T-75 flasks at 37°C and 5 % CO 2 and sub-cultured every 48-72 h by rinsing with Dulbecco's phosphate-buffered saline (DPBSLonza), detaching with trypsin, and quenching by adding the growth medium.…”

“…The location of integrated plasmid DNA into the CHO cell chromosome [11] and silencing of the transgene as a result of epigenetic gene regulation such as DNA methylation [12][13][14] and histone deacetylation [15] are suspected to play a role in instability. In order to overcome these challenges, recent studies have focused on various regulatory elements that can be incorporated into the expression vectors to maintain transgenes in a transcriptionally active state including Locus control regions (LCRs) [16], Scaffold/Matrix Attachment Regions (S/MARs) [17,18] and Ubiquitous Chromatin Opening Elements (UCOEs) [14,[19][20][21]. UCOEs are derived from the heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homologue 3 (HNRPA2B1/CBX3) housekeeping gene locus and are defined as being comprised of an extended methylation-free CpG-island and normally bi-directional promoters that are divergently transcribed [20,22].…”

“…Anti-silencing activity was associated with strongly reduced DNA methylation of promoter, which strongly favours its application in PSC-based cell and gene therapy. In CHO and BHK21 cells, UCOE provided a higher level of transgene expression following stable transfection [19,21,[27][28][29] and maintained the stability of protein expression over 100 generations [30]. In this study, a UCOE vector was used in combination with MTX amplification to improve the frequency of positive clones and to achieve a high volumetric recombinant protein production.…”

Proteolytic Cleavage of Chemerin Protein Is Necessary for Activation to the Active Form, Chem157S, Which Functions as a Signaling Molecule in Glioblastoma

“…Chem163S, Chem158K, Chem157S, and Chem155A-Inclusion of a UCOE in a mammalian expression plasmid allows higher productivity clones to be isolated and grown stably (21,22). We utilized this system to produce different forms of chemerin.…”