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

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    Author
    Lee, Soomin
    Kim, Jeong-Ah
    Kim, Hee-Dae
    Chung, Sooyoung
    Kim, Kyungjin
    Choe, Han Kyoung
    Affiliation
    Univ Arizona, Coll Med Phoenix, Dept Basic Med Sci
    Issue Date
    2019-05
    Keywords
    2A
    expression dynamics
    internal ribosome entry site
    multicistronic elements
    real-time fluorescent imaging
    
    Metadata
    Show full item record
    Publisher
    KOREAN SOC MOLECULAR & CELLULAR BIOLOGY
    Citation
    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 CELLS
    Rights
    © 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 journal
    ISSN
    1016-8478
    EISSN
    0219-1032
    PubMed ID
    31085809
    DOI
    10.14348/molcells.2019.2427
    Version
    Final published version
    Sponsors
    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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