FOREIGN GENE-EXPRESSION (BETA-GALACTOSIDASE) DURING THE CELL-CYCLE PHASES IN RECOMBINANT CHO CELLS

Recombinant mammalian cultures for heterologous gene expression typica lly involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein i n single cells during the cell cycle of Chinese hamster ovary (CHO) ce lls transfected with an expression vector containing the gene for dihy drofolate reductase (dhfr) and the lacZ gene for bacterial beta-galact osidase (a nonsecreted protein). The lacZ gene was under the control o f the constitutive cytomegalovirus promoter. These normally attachment -grown cells were adapted to suspension culture in 10(-7) M methotrexa te, and a dual-laser flow cytometer was used to simultaneously determi ne the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell c ycle phase was evaluated for cells in the exponential growth phase, ea rly plateau phase, and inhibited traverse of the cell cycle during exp onential growth. The results showed that the beta-galactosidase produc tion rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addit ion of aphidicolin, beta-galactosidase content in single cells was hig her than that in exponential phase or plateau phase cells and increase d with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosida se per unit volume of culture was very similar to that in normal expon ential growth. (C) 1993 John Wiley & Sons, Inc.