Gu M B, Kern J A, Todd P, Kompala D S
Department of Chemical Engineering, University of Colorado, Boulder 80309-0424.
Cytotechnology. 1992;9(1-3):237-45. doi: 10.1007/BF02521751.
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.
开展了多项研究以表征基因扩增和外源基因表达对重组中国仓鼠卵巢（CHO）细胞生长的影响。用含有二氢叶酸还原酶（dhfr）基因、人β干扰素（β-IFN）基因或编码β-半乳糖苷酶（β-gal）的lac Z基因的表达载体转染中国仓鼠卵巢（CHO）细胞。这些CHO细胞中的重组基因通过在0、10⁻⁷和10⁻⁶ M甲氨蝶呤（MTX）中生长进行逐步扩增，并且使表达β-gal的细胞适应悬浮培养。采用流式细胞术方法（FCM）来测定细胞群体中扩增的dhfr基因含量和外源β-gal基因表达的分布。还使用了β-gal的生化测定法。发现β-gal表达随着基因扩增的增加而增加。发现在10⁻⁷ M MTX条件下重组CHO细胞的生长速率比无MTX培养基中的重组CHO细胞低20%，并且在10⁻⁶ M MTX条件下的细胞生长速率比在10⁻⁷ M MTX条件下的重组CHO细胞低20%。10⁻⁵ M MTX对CHO-DG44（dhfr⁻）细胞的生长没有影响。因此，重组CHO细胞生长速率的降低被认为主要是由于dhfr和外源基因扩增以及β-半乳糖苷酶表达增加的影响。
