Yuk Inn H Y, Wildt Stefan, Jolicoeur Mario, Wang Daniel I C, Stephanopoulos Gregory
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biotechnol Bioeng. 2002 Jul 5;79(1):74-82. doi: 10.1002/bit.10293.
The growth of anchorage-dependent Chinese hamster ovary (CHO) cells is arrested upon serum deprivation; however, a portion of these cells remain viable for extended time periods in serum-free culture. This work presents a strategy to both rapidly generate a heterogeneous population of CHO cells as well as to select for subpopulations that remain robust and continue to produce recombinant protein when their growth is arrested. Stable expression of recombinant proteins in mammalian cells is often a tedious and time-consuming process because only a small percentage of transfected cells will express sufficient quantities of protein. To overcome the limitations associated with standard transformation and selection methods, bicistronic retroviral expression technology was used. First, bicistronic retroviral constructs encoding for both interferon gamma (IFN-gamma), the model therapeutic protein, and green fluorescent protein (GFP), the quantitative selectable marker, were generated. Next, recombinant retroviruses were obtained from transient transfection of a helper-cell line and were used to infect susceptible CHO cells. Cells with the bicistronic expression module stably integrated into their genome fluoresce green and could thereby be easily isolated by fluorescence-activated cell sorting. Upon subjecting successfully infected cells to serum withdrawal, significant declines in cell viability and GFP expression occurred. After imposing this selection pressure on the cells for 8 days, GFP producers were isolated from the survivors by fluorescence-activated cell sorting and expanded. To evaluate the effectiveness of the screening process, the selected cells were exposed to a second round of serum deprivation. Unlike the original cell population from which it was derived, the subpopulation remained robust and continued to stably express both GFP and IFN-gamma throughout the extended period of serum-free culture. Within 2 weeks, cells selected for recombinant protein production under serum-free conditions were successfully generated and isolated.
依赖贴壁生长的中国仓鼠卵巢（CHO）细胞在血清剥夺后生长停滞；然而，这些细胞中的一部分在无血清培养中仍能长时间保持存活。这项工作提出了一种策略，既能快速产生异质性的CHO细胞群体，又能选择在生长停滞时仍保持活力并继续产生重组蛋白的亚群。在哺乳动物细胞中稳定表达重组蛋白通常是一个繁琐且耗时的过程，因为只有一小部分转染细胞会表达足够量的蛋白。为了克服与标准转化和选择方法相关的局限性，使用了双顺反子逆转录病毒表达技术。首先，构建了编码模型治疗性蛋白干扰素γ（IFN-γ）和定量选择标记绿色荧光蛋白（GFP）的双顺反子逆转录病毒载体。接下来，通过辅助细胞系的瞬时转染获得重组逆转录病毒，并用于感染易感的CHO细胞。双顺反子表达模块稳定整合到基因组中的细胞发出绿色荧光，因此可以通过荧光激活细胞分选轻松分离。对成功感染的细胞进行血清剥夺后，细胞活力和GFP表达显著下降。在对细胞施加这种选择压力8天后，通过荧光激活细胞分选从存活细胞中分离出GFP产生细胞并进行扩增。为了评估筛选过程的有效性，将选定的细胞暴露于第二轮血清剥夺。与原始细胞群体不同，该亚群在无血清培养的延长期间内保持活力，并继续稳定表达GFP和IFN-γ。在2周内，成功产生并分离出了在无血清条件下选择用于重组蛋白生产的细胞。
