• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.118-118
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.118-118
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• 2005

• Journal of fish pathology
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• v.24 no.3
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• pp.297-305
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• 2011

A trap identification system for isolating functional sequences to allow the constitutive expression of foreign protein from Edwardsiella tarda was developed. Using the green fluorescent protein (GFP) reporter-based trap system, various functional sequences to drive heterologous expression of the GFP were selectable in Escherichia coli host. However from the bioinformatic sequence analysis, all the segments predicted as regulatory regions were not native promoters actually existing upstream of endogenous E. tarda genes. Instead, a number of non-authentic sequences, possibly resulted from the random shuffling and/or intermolecular ligation were also proven to be able to display a potent GFP expression in the recombinant E. coli. Further analysis with selected clones showed that both authentic and non-authentic sequences could function in as a constitutive promoter, leading quite a consistent and stable GFP expression after repetitive subcultures. Microscopic examination also confirmed the uniform pattern of GFP expression in every host bacterium. Semi-quantitative assay of GFP showed that there was no clear relationship between expression levels and organizational features of the promoters trapped. Functional promoter-like elements achieved in the present study could be a good starting material for multivalent genetic engineering of E. tarda in order to produce recombinant vaccines in a cost-effective fashion.

• BMB Reports
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• v.43 no.7
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• pp.451-460
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• 2010

Starting with the first publication of lacZ gene fusion in 1980, reporter genes have just entered their fourth decade. Initial studies relied on the simple fusion of a promoter or gene with a particular reporter gene of interest. Such constructs were then used to determine the promoter activity under specific conditions or within a given cell or organ. Although this protocol was, and still is, very effective, current research shows a paradigm shift has occurred in the use of reporter systems. With the advent of innovative cloning and synthetic biology techniques and microfluidic/nanodroplet systems, reporter genes and their proteins are now finding themselves used in increasingly intricate and novel applications. For example, researchers have used fluorescent proteins to study biofilm formation and discovered that microchannels develop within the biofilm. Furthermore, there has recently been a "fusion" of art and science; through the construction of genetic circuits and regulatory systems, researchers are using bacteria to "paint" pictures based upon external stimuli. As such, this review will discuss the past and current trends in reporter gene applications as well as some exciting potential applications and models that are being developed based upon these remarkable proteins.

• Fisheries and Aquatic Sciences
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• v.18 no.2
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• pp.183-193
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• 2015

Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$-actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$-actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.463-468
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• 2003

Polychlorinated biphenyl are toxic pollutants and their degradation is quite slow in the environment. Recently, interest if bioremediation using PCB-degrading bacteria has increaset,. In a previous report, plant terpenes (p-cymene, (S)-(-)-limonene, ${\alpha}-pynene$, and ${\alpha}-terpinene$) have been found to be utilized by a PCB degrader and to induce the biphenyl dioxygenase gene in pure culture. In this study, Pseudomonas pseudoalcaligenes KF707, a PCB-degrading Gram-negative soil bacterium, was used to determine whether the terpene stimulation of PCB degrader occurred in the natural environment. First, P. pseudoalcaligenes KF707 was genetically tagged using a transposon with gfp (green fluorescent protein) as a reporter gone. The population dynamics of P. pseudoalcaligenes KF707 harboring gfp gene in a PCB-contaminated environment was examined with or without terpenoids added to the microcosm. About 10-100-fold increase was found in the population of PCB degraders when terpene was added, compared with control (non-terpenes samples and biphenyl added samples). It was proposed that the gfp-monitoring system is very useful and terpenes enhance the survivability of PCB degraders in PCB-contaminated environments.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.208-211
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• 2000

The recombinant fluorescent chinese hamster ovary (CHO) cell line was developed and optimized through this study for biomonitoring system. This cell line, called KFC-A10, contains recombinant plasmid(pKCFG) constructed in this study for detecting toxic conditions (Mitomicyn C, EDCs, ${\gamma}-ray$, etc.). It is known that c-Fos is involved in proliferation and differentiation of the signal transduction and overexpression of this gene can lead cell to death under the toxic conditions including apoptosis status. Therefore, pKCFG which has the c-fosSRE::GFP is induced by toxic chemicals, especially DNA damage agents and apoptotic chemicals, and produces green fluorescence protein(GFP) under these toxic conditions. Through the characterization of KFC-A10 using fluorescent assays of GFP, it was shown that KFC-A10 cell line had a manifest GFP expression pattern due to various toxicants especially mitomycin C, ${\gamma}-ray$ and bisphenol A. Therefore this study proved the possibility of using GFP as a reporter for detecting various toxicants

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1179-1183
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• 2011

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, p-hydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for p-cresol degradation that is dominantly expressed in colonies.

• Microbiology and Biotechnology Letters
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• v.39 no.4
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• pp.337-344
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• 2011

Integrase MJ1 from the bacteriophage ${\Phi}FC1$ carries out recombination between two DNA sequences (the phage attachment site, attP and the bacterial attachment site, attB) in NIH3T3 mouse cells. In this study, the integration vector containing attP, attB and the integrase gene MJ, was constructed. The integration mediated by integrase MJ1 in Escherichia coli led to excision of LacZ. Therefore, the frequency of integration was measured by the counting of the white colony, which is detectable on X-Gal plates. The extrachromosomal integration in NIH3T3 mouse cells was monitored by the expression of the green fluorescent protein (GFP) as a reporter. To demonstrate integration mediated integrase MJ1 in NIH3T3 cells, vectors containing attP and attB were co-transfected into NIH3T3 cells. The integration was confirmed by fluorescent microscopy. The expression of GFP was induced in NIH3T3 cells expressing MJ1 without accessory factors. By contrast, the excision mediated by the MJ1 between attR and attL had no effect on the expression of GFP. These results suggest that integrase MJ1 may enable a variety of genomic modifications for research and therapeutic purposes in higher living cells.

• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1790-1798
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• 2019

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'-end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28℃ but weak at 30℃. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.