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

Berta Capella Roca,Nga Lao,Niall Barron,Padraig Doolan,Martin Clynes

doi:10.1074/jbc.ra119.011162

Abstract

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.

Highlights

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
To assess which of the two reactions was causing the putrescine-dependent phenotype observed, Chinese hamster ovary (CHO)-K1 cells were cultured for fourteen passages in three conditions: (i) serum-free media (SFM)-F12 medium lacking putrescine, (ii) SFM-F12 medium supplemented with L-ornithine (10 ␮M), and (iii) SFM-F12 control medium
It is noteworthy that CHO-K1 cells were grown in SFM-F12 medium supplemented with putrescine prior to the start of the SFM-F10 test, with no previous adaptation to SFM-F10 medium

Summary

Introduction

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. Single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which displayed stable expression and healthy growth. 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. Stable cell line development and clonal selection still remain as a costly, laborand time-consuming step [3]

Objectives

Results

Conclusion