• Fisheries and Aquatic Sciences
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• v.19 no.9
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• pp.41.1-41.7
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• 2016

Background: The transgenic approach using estrogen-responsive regulator in fish has been given much attention as a potential means to detect and/or address estrogen-related aquatic pollutions. In order to address the development stage- and reproduction status-dependent expression patterns of the chgH-rfp transgene (red fluorescent protein transgene driven by choriogenin H promoter) in marine medaka Oryzias dancena, naturally occurring red fluorescent protein (RFP) signals under non-exposed conditions as well as the transgenically induced RFP signals under estrogen-exposed conditions were assayed. Results: Female transgenics begun to show naturally occurring RFP signals from the age of 7 weeks post hatching (WPH) without experimental estrogen exposure. Afterward, these RFP signals in female transgenics became robust with the progress of ovarian maturation. On the other hand, male transgenics did not show any naturally occurring RFP signal under non-exposed conditions irrespective of developmental stages and maturation statue. Upon exposures using estradiol-$17{\beta}$ (E2) and $17{\alpha}$-ethinylestradiol (EE2), RFP signals were significantly induced specifically in the livers of transgenic males. Conclusions: Male chgH-rfp transgenics were able to keep the "off" state of RFP expression during their entire life cycle unless exposed to exogenous estrogens. Owing to their tight regulation capability of estrogen-responsive transgene, transgenesis of chgH-rfp in male marine medaka could offer a useful model system for future ecotoxicogenomic studies regarding estrogenicity-related issues in aquatic and marine environments.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.65-71
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• 2015

The functional utility of a transgenic marine medaka Oryzias dancena strain carrying the red fluorescent protein (RFP) gene driven by an endogenous choriogenin H (chgH) promoter was evaluated for its ability to detect waterborne $17{\alpha}$-ethinylestradiol (EE2), a synthetic estrogen derivative. The chgH:rfp transgenic marine medaka larvae showed an age-dependent tendency in the efficiency of EE2-mediated transgene expression, in which transgenic larvae older than 6 days post-hatching displayed a more effective response in their transgene expression to EE2 than did younger hatchlings. During experimental exposures to high concentrations of EE2 (200 to 1,000 ng/L), the transgenic responses in the hatchlings were broadly dose- and duration-dependent. With exposures using lower doses of EE2 (25, 50 and 100 ng/L), EE2-induced transgenic RFP was also observed in the transgenic larvae, although the lower doses required exposure of longer duration. Under the EE2 exposure and microscope assay conditions used in our study, transgenic marine medaka larvae exhibited a similar degree of EE2-mediated RFP phenotype expression at various salinity levels (0, 15 and 30 ppt).

• Fisheries and Aquatic Sciences
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• v.15 no.4
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• pp.317-324
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• 2012

We characterized the cytoskeletal beta-actin (${\beta}$-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.

• Molecules and Cells
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• v.23 no.3
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• pp.357-362
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• 2007

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Analytical Science and Technology
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• v.26 no.1
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• pp.106-112
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• 2013

PDE plays an important role in cAMP-mediated cellular signaling within the cells. The proper targeting of each PDE is mediated by unique N-terminal of each PDE isoform. It has been recently reported that supershort-, short- and long-forms of PDE4 in Aplysia were cloned in Aplysia. Long-form of ApPDE4 was localized at plasma membrane and presynaptic terminal in Aplysia sensory neurons. However, it remains elusive which part of ApPDE4 is minimal region for the proper targeting and what are the effects on the cell functions. Here, we identified that N-terminal 13 amino acids of ApPDE4 long-form is minimal regions for the plasma membrane targeting. In addition, overexpression of ApPDE4(N20)-mRFP could induce morphological changes in HEK293T cells. Interestingly, mRFP-$PLC{\delta}1$(PH), which selectively binds to PI4,$5P_2$, could induce morphological changes in similar with that by ApPDE4(N20)-mRFP. These results suggested that binding of ApPDE4(N20) to lipids including PI4,$5P_2$ might be responsible for targeting of ApPDE4 to plasma membrane and morphological changes in HEK293T cells.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.81-85
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• 2007

Glucocorticoid hormone regulates numerous physiological processes, such as regulation of metabolism, and anti-inflammatory and immunosuppressive actions via the activation and repression of gene expression. Here we described a lentivirus-based reporter vector system expressing red fluorescent protein (mRFP) or firefly luciferase (Luc) under the control of a glucocorticoid-responsive element that allows observation of the temporospatial pattern of glucocorticoid induced GR-mediated signaling on a cellular level. Moreover, usage of the chromatin insulator of the chicken ${\beta}$-globin locus induced a marked increase of sensitivity of glucocorticoid inducible promoter of a reporter gene. Use of this method will be applicable of screening for agonist and antagonist of GR in vitro, and also a reporter gene assay for the in vivo determination of the GR-mediated gene activation.

• Fisheries and Aquatic Sciences
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• v.15 no.4
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• pp.325-335
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• 2012

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

• Molecules and Cells
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• v.37 no.3
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• pp.248-256
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is a N-acetylhexosamine kinase that belong to the sugar kinase/heat shock protein 70/actin superfamily. In this study, we investigated both the expression and function of NAGK in neurons. Immunohistochemistry of rat brain sections showed that NAGK was expressed at high levels in neurons but at low levels in astrocytes. Immunocytochemistry of rat hippocampal dissociate cultures confirmed these findings and showed that NAGK was also expressed at low levels in oligodendrocytes. Furthermore, several NAGK clusters were observed in the nucleoplasm of both neuron and glia. The overexpression of EGFP- or RFP (DsRed2)-tagged NAGK in rat hippocampal neurons (DIV 5-9) increased the complexity of dendritic architecture by increasing the numbers of primary dendrites and dendritic branches. In contrast, knockdown of NAGK by shRNA resulted in dendrite degeneration, and this was prevented by the co-expression of RFP-tagged NAGK. These results suggest that the upregulation of dendritic complexity is a non-canonical function of NAGK.

• Development and Reproduction
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• v.23 no.1
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• pp.55-61
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• 2019

To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.