ISSN:
1573-0778
Keywords:
CMV promoter
;
dhfr
;
foreign gene expression
;
gene amplification
;
growth rate reduction
;
metabolic burden
;
recombinant CHO cells
;
SV40 promoter
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Abstract Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo r ) and thelacZ gene which codes for intracellular β-galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDβG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and β-galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo r gene expression, must be negligible, as higher expression of β-galactosidase (1.5×10−6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10−7 units/cell). Thus, the main factor causing severe growth reduction (“metabolic burden”) in cells containing the amplifieddhfr gene system was not overexpression of β-galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00767763
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