Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, MN, 55455-0312, USA.
Cytotechnology. 2006 Sep;52(1):13-24. doi: 10.1007/s10616-006-9020-9. Epub 2006 Nov 22.
Transient gene expression is frequently used in industry to rapidly generate usable quantities of a protein from cultured cells. In gene therapy applications it is used to express a therapeutic protein in vivo. A quantitative assessment of the expression kinetics is important because it enables optimization and control of culture conditions for higher productivity. Previous experimental studies show a characteristic peak in average protein expression per cell after transfection followed by an exponential decrease of the expressed protein. Here, we show that the exponential decrease in single cell expression of enhanced Green Fluorescent Protein (eGfp) occurs in discrete steps. We attribute this to the absence of plasmid replication and to symmetric partitioning of plasmid and eGfp between dividing cells. This is reflected in the total eGfp in the bioreactor, which increased at a constant rate throughout the experiment. Additionally, the data provide a detailed time course of cell physiology during recovery from electroporation. The time course of cell physiology precisely indicates when the culture shifts growth phases. Furthermore, the data indicate two unique stationary phases. One type of stationary phase occurs when proliferation ceases while cells decrease their cell size, maintain granularity, and mean eGfp content decreases. The second type occurs when proliferation ceases while cells increase their cell size, increase granularity, and surprisingly maintain eGfp content. The collected data demonstrate the utility of automated flow cytometry for unique bioreactor monitoring and control capabilities in accordance with the US Food and Drug Administration's Process Analytical Technology initiative.
瞬时基因表达通常用于工业领域，以从培养细胞中快速产生大量可用的蛋白质。在基因治疗应用中，它用于在体内表达治疗性蛋白质。对表达动力学进行定量评估非常重要，因为它可以优化和控制培养条件以提高生产力。先前的实验研究表明，转染后每个细胞的平均蛋白质表达会出现特征性峰值，随后表达的蛋白质呈指数下降。在这里，我们表明，增强型绿色荧光蛋白（eGFP）在单细胞表达中的指数下降是分阶段发生的。我们将其归因于质粒复制的缺失以及质粒和 eGFP 在分裂细胞之间的对称分配。这反映在生物反应器中的总 eGFP 上，整个实验过程中 eGFP 以恒定速率增加。此外，这些数据提供了电穿孔恢复过程中细胞生理学的详细时间过程。细胞生理学的时间过程准确地指示了培养物何时进入生长阶段的转变。此外，数据还表明存在两种独特的稳定期。一种类型的稳定期发生在增殖停止而细胞减小细胞大小、保持颗粒度且平均 eGFP 含量降低时。第二种类型发生在增殖停止而细胞增大细胞大小、增加颗粒度且令人惊讶的是保持 eGFP 含量时。收集的数据表明，自动流式细胞术具有独特的生物反应器监测和控制能力，符合美国食品和药物管理局的过程分析技术倡议。
