• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.127-140
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• 2019

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

• Molecules and Cells
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• v.42 no.5
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• pp.418-425
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• 2019

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.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.87-87
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• 2002

Previously, we observed high level expression of goat β-casein/genomic hGH fusion gene in mammary gland of transgenic mice. To develop an expression vector to make a human granulocyte-colony stimulating factor (hG-CSF) protein efficiently produced in milk of transgenic animals, we designed a new bicistronic vector using the goat β-casein/genomic hGH fusion gene as regulation sequences for expression and internal ribosome entry site (IRES) as a mediator for second gene expression. This vector was constructed by insertion of encephalomyocarditis virus (EMCV) IRES-dependent second gene region coupled with hG-CSF cDNA into 3' untranslated region of an intact hGH gene. By microinjcetion, four transgenic mice were generated and three of them transmitted the bicistronic vector to their progeny. (omitted)

• Development and Reproduction
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• v.14 no.1
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• pp.13-19
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• 2010

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

• Korean Journal of Microbiology
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• v.35 no.1
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• pp.28-34
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• 1999

Fragmentation vectors are used to analyze function and genomic structure of a gene of interest by creating deletion derivatives of large fragments of genomic DNA cloned as yeast artificial chromosomes (YACs). Herein, we developed a new hicistronic fragmentation vector that contains internal ribosomal entry sile (IRES) of encephalomyocarditis vin~s (EMCV) and $\beta$-galactosidase as a reporter gene. This vector system provides a novcl loo1 to analyze expression patterns of a gene of interest due to simultaneous expression of a target gene as well as $\beta$-galactosidase driven from a single message. In addition, the bicistronic fragmentation vector contains four rare-cutting restriction enzyme sites in the polycloning sites which can be used to conveniently insert any kinds of genes and therefore facilitates targeting DNA scgments into YAC by means of homologous recombination. This approach establishes a paradigm for manipulation of mammalian DNA segments and characterization of expression and regulatory regions of mammalian gene cloned as YAC.