Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcar...Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.展开更多
Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synth...Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.展开更多
目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂...目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。展开更多
文摘Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.
文摘Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.
文摘目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。
