Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea.
Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
Mol Cells. 2019 May 31;42(5):418-425. doi: 10.14348/molcells.2019.2427.
Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.
多顺反子元件，如内部核糖体进入位点（IRES）和 2A 样切割序列，在外源基因的真核异位表达中起着至关重要的作用。为了利用多顺反子元件，已经研究了多顺反子载体中元件的切割效率和顺序；然而，多顺反子元件介导的表达动力学仍然不清楚。在这里，我们研究了脑炎心肌炎病毒（EMCV）IRES 和猪肠道病毒 1 2A（p2A）介导的表达动力学。通过利用每分钟分辨率的实时荧光成像，我们监测了由 EMCV IRES 或 p2A 桥接的荧光报告基因在两个独立的培养细胞系 HEK293 和 Neuro2a 中的表达。我们观察到两种多顺反子元件中的两个荧光报告基因之间存在显著的相关性，p2A 在 HEK293 中的相关系数更高，但在 Neuro2a 中 IRES 介导的表达和 p2A 介导的表达的相关系数相似。我们进一步通过会聚交叉映射（CCM）分析多顺反子元件的因果关系。CCM 表明，在所检查的所有四种条件下，前一个基因的表达都对随后基因的动力学产生因果影响。与交叉相关一样，在 HEK293 中，p2A 的预测能力高于 IRES，而在 Neuro2a 中，两种多顺反子元件的预测能力则无法区分。总之，我们根据简单的双顺反子载体和实时荧光监测报告了 EMCV IRES 和 p2A 介导的表达均具有显著的时间相关性。该系统还为在各种生理条件下研究不同多顺反子元件介导的表达的动态方面提供了一个有价值的平台。
