Department of Chemical Engineering, University of Colorado, Boulder, CO, 80309, USA.
Cytotechnology. 2002 Jan;38(1-3):23-35. doi: 10.1023/A:1021125208710.
Expression levels of reporter protein driven by Mouse Mammary Tumor Virus Promoter system were improved by expressing its specific transcription factor (glucocorticoid receptor) from a different expression vector. The vector that expresses glucocorticoid receptor (GR) also contained dihydrofolate reductase (dhfr) gene as a selection marker. In the presentstudy we amplified the glucocorticoid receptor gene (gr)along with the dhfr gene by adapting the cell lines to increasing concentrations of methotrexate, an antifolate analog. Stepwise increases in the volumetric titers of a secreted reporter glycoprotein, Secreted Alkaline Phosphatase (SEAP), were observed in recombinant Chinese hamster ovary (CHO) cellsgrowing in increased concentrations of methotrexate. Western andRT-PCR analysis showed that this increase in volumetric titers is associated with higher levels of GR expressed in CHO cellsgrowing in increased concentration of methotrexate. A stablytransfected cell line growing in 10(-6) M methotrexate wasgrown in suspension culture and induced with 10(-7) Mdexamethasone. The SEAP volumetric titers reached a peak of approximately 23 mug ml(-1) on the 5th day after induction.Inducing these cells with increasing concentrations of dexamethasone resulted in increased specific productivity. These high volumetric productivities were further increased in fed-batch bioreactors.
通过在不同的表达载体中表达其特定的转录因子（糖皮质激素受体），可以提高由鼠乳腺肿瘤病毒启动子系统驱动的报告蛋白的表达水平。表达糖皮质激素受体 (GR) 的载体还包含二氢叶酸还原酶 (dhfr) 基因作为选择标记。在本研究中，我们通过使细胞系适应越来越高浓度的氨甲喋呤（一种抗叶酸类似物），扩增了糖皮质激素受体基因 (gr) 和 dhfr 基因。在越来越高浓度的氨甲喋呤中生长的重组中国仓鼠卵巢 (CHO) 细胞中，观察到分泌型碱性磷酸酶 (SEAP) 的分泌报告蛋白的体积效价逐渐增加。Western 和 RT-PCR 分析表明，CHO 细胞中 GR 的表达水平升高与体积效价的增加有关，这些 CHO 细胞在越来越高浓度的氨甲喋呤中生长。在 10(-6) M 氨甲喋呤中生长的稳定转染细胞系在悬浮培养中生长并以 10(-7) M 地塞米松诱导。诱导后第 5 天，SEAP 体积效价达到约 23 mug ml(-1)的峰值。用越来越高浓度的地塞米松诱导这些细胞会导致特异性产率增加。在分批补料生物反应器中进一步提高了这些高体积产率。
