X. Mazur et al., Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27, BIOTECH PR, 14(5), 1998, pp. 705-713
We constructed stable Chinese hamster ovary (CHO) cell lines which conditio
nally and coordinately express the model product gene secreted alkaline pho
sphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p5
3 mutant deficient in apoptotic but not cell-cycle arrest function. The use
of dicistronic expression technology allowed the conditional expression of
the model product gene and the cytostatic gene in a coordinated fashion fr
om a single expression unit under the control of the tetracycline-responsiv
e promoter PhCMV*-1. Due to the presence of a cytostatic gene in the multic
istronic expression unit, the growth behavior of the engineered CHO cell li
nes could be controlled by the addition or withdrawal of the exogenous agen
t tetracycline to or from the cell culture medium. Withdrawal of tetracycli
ne resulted in sustained growth arrest of the stable cell lines for a prolo
nged period. The growth arrest of such cell lines was found to be accompani
ed by a 10-15-fold increase in their production of SEAP per cell. This cont
rolled proliferation technology allows the design of a novel two-stage prod
uction process which consists of a proliferation phase leading to the desir
ed cell density, followed by an extended production phase during which the
cells remain growth-arrested and increase cell-specific production of a het
erologous protein.