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Mol Cells. 2019 May 31;42(5):418-425. doi: 10.14348/molcells.2019.2427.

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence.

Author information

1
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea.
2
Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
3
Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004, USA.
4
Department of Brain and Cognitive Sciences, Scranton College, Ehwa Womans University, Seoul 03760, Korea.
5
Korea Brain Research Institute (KBRI), Daegu 41062, Korea.

Abstract

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.

KEYWORDS:

2A; expression dynamics; internal ribosome entry site; multicistronic elements; real-time fluorescent imaging

PMID:
31085809
DOI:
10.14348/molcells.2019.2427
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