METABOLIC BURDEN IN RECOMBINANT CHO CELLS - EFFECT OF DHFR GENE AMPLIFICATION AND LACZ EXPRESSION

Foreign protein production levels in two recombinant Chinese hamster o vary (CHO) cell lines were compared in cells transfected with differen t expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo(r)) and the lacZ gene which codes for intracellular be ta-galactosidase, with both genes controlled by the constitutive simia n virus (SV40) promoter. The other vector CD beta G contained the ampl ifiable dhfr gene and lacZ gene, controlled by the constitutive SV40 a nd cytomegalovirus (CMV) promoters, respectively. Cell growth and beta -galactosidase expression were compared quantitatively after cells wer e selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred i n recombinant CHO cells in which the lacZ and dhfr genes were highly a mplified and expressed. In contrast, the combined effects of the unamp lified neo(r) gene and lacZ gene expression on the growth kinetics wer e small. Any metabolic burden caused by lacZ gene expression, which wa s evaluated separately from the effect of neo(r) gene expression, must be negligible, as higher expression of beta-galactosidase (1.5 x 10(- 6) units/cell) occurred in unamplified cells compared to the cells in which lacZ was amplified by the dhfr-containing vector (3 x 10(-7) uni ts/cell). Thus, the main factor causing severe growth reduction (''met abolic burden'') in cells containing the amplified dhfr gene system wa s not overexpression of beta-galactosidase but dhfr and lacZ gene co-a mplification and dhfr gene expression.