Mol. Cells 2019; 42(3): 245  https://doi.org/10.14348/molcells.2019.2427
Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence
Soomin Lee1, Jeong-Ah Kim1,2, Hee-Dae Kim3, Sooyoung Chung4, Kyungjin Kim1, and Han Kyoung Choe1,5,*
1Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea, 2Department of Biological Sciences, Seoul National University, Seoul 08826, Korea, 3Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004, USA, 4Department of Brain and Cognitive Sciences, Scranton College, Ehwa Womans University, Seoul 03760, Korea, 5Korea Brain Research Institute (KBRI), Daegu 41062, Korea
Received November 19, 2018; Revised March 30, 2019; Accepted April 5, 2019.; Published online April 26, 2019.
© Korean Society for Molecular and Cellular Biology. All rights reserved.

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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 imagin


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30 April 2019 Volume 42,
Number 4, pp. 285~377

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