C. GFP for a total of 42 decades. By using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human being erythropoietin (hEPO), which also displayed stable manifestation and healthy growth. The hEPO-expressing clones grew in commercial press, such as BalanCD and CHO-S serum-free press (SFM)CII, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 decades), with a minimal decrease in hEPO titer by the end of the tradition. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and additional mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have founded an easy-to-apply option selection system that effectively produces mammalian cell clones expressing biopharmaceutically relevant or additional recombinant proteins without the need for any harmful selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity. and and and (19) reported a lack of arginase activity inside a CHO-K1 cell collection in serum-free conditions, resulting in a polyamine-dependent phenotype. In accordance with their findings, it was hypothesized that a lack of arginase expression could be also happening in our parental cell collection. Based RU 24969 on that and, to exploit this phenotype, we designed a selection system for CHO-K1 maker cell lines. As a proof of concept, a commercial arginase-expressing vector, pcDNA3.1-mArg (Addgene), was first transfected and determined in media missing putrescine (NoP) compared with media with putrescine (P). A negative control, consisting of WT untransfected cells, was also included. CHO-K1 cells transfected and selected in SFM-F12 medium lacking putrescine resumed cellular growth by passage 6 (day time 20), finally reaching VCD and viability profiles similar to the parental control cells in putrescine-containing press by passage 8 (day 26) (Fig. 2, and and and and and arginase RQ profiles were observed. Interestingly, from passage 4 (12 generations), an increase in the RQ of both and Rabbit polyclonal to DUSP14 arginase were observed with all clones except clone 11. This effect might be related to the transference of clones from putrescine-containing medium (during single-cell cloning) to SFM-F12 medium depleted of putrescine, increased stringency of selection. Open in a separate window Physique 4. Long-term growth and stable expression of isolated GFP-expressing clones in selective medium. and arginase mRNA expression of isolated GFP-expressing clones in selective medium. Relative quantification (and arginase mRNA levels in seven GFP-expressing clones growing in selective medium for 14 passages (42 generations) was normalized to the mRNA levels in passage 2. The endogenous gene was used RU 24969 to standardize the results. Single clones stably expressing a therapeutically relevant recombinant protein can be generated in polyamine and l-ornithine-free media To RU 24969 confirm whether the selection system designed would support the generation and preferential survival of clones expressing pharmaceutically relevant therapeutics, a bicistronic vector expressing human erythropoietin (and arginase mRNA expression relative to passage 2 was performed on three phenotypically divergent clones: clone 4 (low producer but fast growing), clone 10 (high producer but moderate growth), and clone 18 (medium producer with moderate growth). Both clones 10 and 18 were found to be stable, displaying levels of expression similar to those in passage 2 for at least 36 generations (P12) (Fig. 8). In contrast, the low RU 24969 producer clone 4 displayed a 20% decrease on mRNA expression at 18 generations (P6), dropping to less than 50% relative to the expression at passage 2 by passage 8. Nonetheless, both and arginase expression were detected over the 42 generations (P14) (Fig. 7). Open in a separate window Physique 6. hEPO-expressing populations can be isolated in polyamine-free medium. and and arginase mRNA expression of isolated hEPO-expressing clones in selective medium. Relative quantification (and.

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