Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence
Affiliation
Univ Arizona, Coll Med Phoenix, Dept Basic Med SciIssue Date
2019-05Keywords
2Aexpression dynamics
internal ribosome entry site
multicistronic elements
real-time fluorescent imaging
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KOREAN SOC MOLECULAR & CELLULAR BIOLOGYCitation
Lee, S., Kim, J. A., Kim, H. D., Chung, S., Kim, K., & Choe, H. K. (2019). Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence. Molecules and cells, 42(5), 418.Journal
MOLECULES AND CELLSRights
© The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.Collection Information
This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.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.Note
Open access journalISSN
1016-8478EISSN
0219-1032PubMed ID
31085809Version
Final published versionSponsors
National Research Foundation of Korea (NRF) of the Ministry of Science and ICT [NRF-2017R1C1B2008775, NRF-2017R1A4A1015534, NRF-2018M3C7A1022310]; KBRI basic research program through Korea Brain Research Institute - Ministry of Science and ICT [17-BR-04]ae974a485f413a2113503eed53cd6c53
10.14348/molcells.2019.2427
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