Fakultät für Physik, Ludwig-Maximilians-Universität, Geschwister-Scholl-Platz 1, D-80539 München, Germany.
Biotechnol Bioeng. 2010 Mar 1;105(4):805-13. doi: 10.1002/bit.22604.
In non-viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine- and PEI-mediated exogenous gene expression distributions from single cell time-lapse analysis. Broad Poisson-like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co-transfection analysis with YFP- and CFP-coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors.
在非病毒基因传递中，转基因表达的差异源于成功转染的质粒数量较少。在这里，我们通过单细胞时程分析实验确定了脂质体和聚乙烯亚胺介导的外源基因表达分布。当与同步细胞系一起使用时，两种转染试剂均观察到稳态表达的广泛泊松样分布。同时，用 YFP 和 CFP 编码质粒进行共转染分析表明，多个质粒同时表达，表明质粒以相关单位（复合物）同时传递。我们提出了一种转染的数学模型，其中假设存在一个随机的两步过程，第一步是复合物进入细胞核的低概率进入步骤，随后是从传递的复合物中释放和激活少量质粒。这个概念上简单的模型一致地预测了观察到的转染细胞的比例、共转染比率和表达水平分布。它给出了每个复合物的有效质粒数量，并阐明了相关噪声的来源，从而为评估和改进非病毒载体提供了一个平台。
