• 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.

• 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)

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.772-777
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• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• Journal of fish pathology
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• v.34 no.1
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• pp.17-22
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• 2021

Eukaryotic translation is initiated by either cap-dependent or cap-independent way, and the cap-independent translation can be initiated by the internal ribosomal entry site (IRES). In this study, to know whether the 5'UTR leader sequence of marine birnavirus (MABV) segment A and segment B can act as IRES, bicistronic vectors harboring a CMV promoter-driven red fluorescent gene (mCherry) and poliovirus IRES- or MABV's leader sequence-driven green fluorescent gene (eGFP) were constructed, then, transfected into a mammalian cell line (BHK-21 cells) and a fish cell line (CHSE-214 cells). The results showed that the poliovirus IRES worked well in BHK-21 cells, but did not work in CHSE-214 cells. In the evaluation of MABV's leader sequences, the reporter eGFP gene under the 5'UTR leader sequence of MABV's segment A was well-translated in CHSE-214 cells, indicating 5'UTR of MABV's segment A initiates translation in the cap-independent way and can be used as a fish-specific IRES system. However, the 5'UTR leader sequence of MABV's segment B did not initiate translation in CHSE-214 cells. As the precise mechanism of birnavirid IRES-mediated translation is not known, more elaborate investigations are needed to uncover why the leader sequence of segment B could not initiate translation in the present study. In addition, further studies on the host species range of MABV's segment A IRES and on the screening of other fish-specific IRESs are needed.

• 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 Korean Society of Toxicology Conference
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• 2001.10a
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• pp.189-189
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• 2001

For a continuous expression of human cytochrome p450 3A5 (CYP3A5) and NADPH-cytochrome P450 reductase (CYPR) proteins, bicistronic construct (CYP3A5BC-LNCX2) was made using internal ribosome entry site (IRES). As for mammalian cell expression, we used pLNCX2 retroviral vector; and using calcium phosphate, plasmid transfer was achieved in 293GPG cell and transduced in Chinese hamster ovary (CHO) cell.(omitted)

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.234-241
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• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.