Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.
Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.
Gu
M B
MB
Department of Chemical Engineering, University of Colorado, Boulder 80309-0424.
Kern
J A
JA
Todd
P
P
Kompala
D S
DS
D000818:Animals; D016466:CHO Cells / Q000201:enzymology / Q000502:physiology*; D002452:Cell Count; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D005784:Gene Amplification / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis*
D011994:Recombinant Proteins; D001616:beta-Galactosidase; D008727:Methotrexate