Altered gene expression in CHO cells following polyamine starvation.

AIM: To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). CONCLUSIONS: Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.

Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells.

While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (-FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5-5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to -FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested.

An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals.

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

Zinc supplementation increases protein titer of recombinant CHO cells.

In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM + A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM + A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3 × 106 cells/ml vs 4.2 × 106 cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM + A (2.2 × 106 cells/ml vs 3.2 × 106 cells/ml, day 5). Supplementation with copper at 13.7-20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures.

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance.

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.