Novel surface tagging technology for selection of complex proliferation-controlled mammalian cell phenotypes

Regulated overexpression of the cyclin dependent kinase inhibitor p27 enables biphasic production processes which consist of a nonproducing expansion phase followed by an extended proliferation-arrested production phase. During the growth-arrested production phase proliferation-competent mutants emerge as a consequence of genetic drift and strong counterselection. Here, we evaluate the use of cell surface markers for ex vivo selection of growth-arrested phenotypes by magnetic or FACS-mediated cell sorting. Multigene metabolic engineering resulted in a Chinese hamster ovary- (CHO) derived cell line CHO-SS101(5), which expresses the model product protein SEAP (secreted alkaline phosphatase), the human cyclindependent kinase inhibitor p27, and a membrane-anchored multidomain surface marker Hook in a tricistronic tetracycline-repressible manner. In the absence of tetracycline in the cell culture medium, p27 mediated a G1-phase-specific cell-cycle arrest of CHO-SS101(5) and resulted in a fivefold increase in SEAP production compared to proliferation-competent control cells. Concomitant expression of Hook enabled FACS- or magnetic-based selection of CHO-SS101(5) cells from various mixed populations. Surface selection of engineered cells will likely become important for biopharmaceutical manufacturing and for in vivo maintenance of treated cells in gene therapy and tissue engineering.