Abstract
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 β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-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 β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-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 ofdhfr and foreign gene amplification and increased β-galactosidase expression.
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Abbreviations
- Ampr :
-
ampicillin resitance gene
- CDβG:
-
plasmid bearingdhfr andlac Z genes
- CMV:
-
cytomegalovirus promoter
- CHO:
-
Chinese hamster ovary
- dhfr :
-
dihydrofolate reductase gene
- DHFR:
-
dihydrofolate reductase enzyme
- dFBS:
-
dialyzed fetal bovine serum
- FBS:
-
fetal bovine serum
- FCM:
-
flow cytometric method
- FITC-MTX:
-
fluorescein-methotrexate
- β-gal:
-
β-galactosidase (E.C. 3.2.1.23)
- β-IFN:
-
human β-interferon
- Met IIA :
-
metallothionein IIA promoter
- MTX:
-
methotrexate (4-amino-10-methylfolic acid)
- pSVdMIF:
-
plasmid bearingdhfr and β-IFN genes
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Gu, M.B., Kern, J.A., Todd, P. et al. Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells. Cytotechnology 9, 237–245 (1992). https://doi.org/10.1007/BF02521751
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DOI: https://doi.org/10.1007/BF02521751