ISSN:
0006-3592
Keywords:
cell cycle analysis
;
β-galactosidase
;
gene expression, foreign
;
dihydrofolate reductase
;
recombinant CHO cells
;
cytomegalovirus promoter
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial β-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10-7 M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (β-galactosidase) content of single living cells. Expression of β-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the β-galactosidase production 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 addition of aphidicolin, β-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of β-galactosidase per unit volume of culture was very similar to that in normal exponential growth. © 1993 John Wiley & Sons, Inc.
Additional Material:
11 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260420914
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