Key result
The p2A cleaving sequence provided a higher temporal correlation between two fluorescent reporters compared to the EMCV IRES sequence in HEK293 cells, while both performed similarly in Neuro2a cells.
Why the study?
While cleavage efficiency and element order in multicistronic vectors have been investigated, the dynamics of multicistronic element-mediated expression remained unclear.
This study demonstrates significant temporal correlation and causal relationships in bicistronic expression mediated by IRES and p2A, providing a platform to examine dynamic expression aspects in eukaryotic cells.
Answers are grounded in this paper and cite their sources.
Cell studies offer no clinical guidance yet; leaves open optimal bicistronic vector design for dynamic expression research.
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.
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Lee et al. (2019) studied this question. p2A cleaving sequence vs. EMCV IRES sequence was evaluated on Temporal correlation between tGFP and mCherry fluorescent signals (R2 = 0.5606 for p2A vs 0.4044 for IRES in HEK293, p=<0.001). The p2A cleaving sequence provided a higher temporal correlation between two fluorescent reporters compared to the EMCV IRES sequence in HEK293 cells, while both performed similarly in Neuro2a cells.
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