• Korean Journal of Microbiology
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• v.42 no.2
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• pp.142-148
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• 2006

In order to develop chitosanase for the production of chitosan oligosaccharides, a chitosanase-producing bacterium was isolated from the traditional fermented soybean, Meju, and identified as Bacillus amyloliquefaciene MJ-1. The cloned chitosanase gene, 825 bp in size, encoded a single peptide of 274 amino acids with a estimated molecular mass of 30.9 kDa. The deduced amino acid sequence showed significant homology with microbial chitosanases. The recombinant chitosanase was expressed in Escherichia coli upon induction with isopropyl-D-thiogalactopyranoside, and purified using $Ni^{2+}-NTA$ agarose column chromatography. The maximal activity of the recombinant chitosanase is at pH 5.0 and $60^{\circ}C$. The recombinant chitosanase is stable between pH 5.0 and pH 7.0 at $37^{\circ}C$ for 30 min, and more than 75% of the activity still remain at $80^{\circ}C$ for 30 min incubation.

• Korean Journal of Poultry Science
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• v.48 no.3
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• pp.111-122
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• 2021

Recently, interleukin 34 (IL-34) was identified as the second functional ligand for macrophage colony-stimulating factor receptor (M-CSFR). IL-34 functions similarly to M-CSF through its binding to the M-CSFR. There is still insufficient information on IL-34 in chickens, which has until now been reported only through predicted sequences and not through experimental research. Thus, to confirm its expression and to determine its potent biological activity, several chicken lines and cell lines were used. Cloning of recombinant chicken IL-34 and M-CSF genes was performed to investigate their modulatory effects on proinflammatory cytokine expression in vitro. The expression levels of IL-34, M-CSF, and M-CSFR genes were upregulated in broiler chickens with leg dysfunction (cause unknown). However, IL-34 was downregulated in most pathogen-stimulated tissues. M-CSFR expression was enhanced by recombinant IL-34 and M-CSF proteins in vitro. IFN-γ expression was enhanced by recombinant IL-34, but not by M-CSF. However, IL-12 expression was not regulated in any of the treated cells, and IL-1β was decreased in all tissues. These results indicate that IL-34 and M-CSF have roles in both the classical and alternative macrophage activation pathways. Collectively, our findings demonstrate the expression of IL-34 in chickens for pathogenic trials, both in vitro and in vivo. Our results suggest that the IL-34 protein plays a role in both pro- and anti-inflammatory functions in macrophages. Therefore, further research is needed to determine the cytokines or chemokines that can be induced by IL-34 and to further elucidate the functions of IL-34 in the inflammatory pathway.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.255-264
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• 2018

This experiment was conducted to analyse the effects of flavone supplementation on the preimplantation development of in-vitro produced porcine embryos. During in-vitro development, immature oocytes and early embryos were exposed to different concentrations of flavone (0, $1{\mu}M$, $25{\mu}M$, $50{\mu}M$, and $100{\mu}M$ respectively). Results showed that $100{\mu}M$ of flavone significantly reduced the intracellular ROS levels of oocytes accompanied with a significant rise in GSH level. In parthenogenesis, no significant change was observed in the cleavage rates whether flavone was supplemented in IVM or IVC media. In IVM supplemented group, the blastocyst development rate was significantly enhanced by $1{\mu}M$ concentration than other groups (51.5% vs. 41.3%, 44.0%, 36.3%, 31.7%; P<0.05) respectively. However, in IVC group $1{\mu}M$ concentration significantly improved the blastocysts production than $50{\mu}M$ and control groups (50.0% vs. 40.5%, 38.0%; P<0.05) respectively. Following nuclear transfer, the cleavage rate of IVM group was significantly more in $1{\mu}M$ than $50{\mu}M$ and $100{\mu}M$ groups (92.9% vs. 89.7%, 87.8%; P<0.05), followed by similar pattern of cloned blastocysts production being significantly higher in $1{\mu}M$ group than $50{\mu}M$, $100{\mu}M$ and control groups (16.8% vs. 9.0%, 7.1%, 12.8%; P<0.05) respectively. In IVC group, $1{\mu}M$ concentration resulted in significantly higher cleavage rate than $25{\mu}M$ and $50{\mu}M$ groups (91.7% vs. 87.8%, 88.8%; P<0.05) respectively. However, the blastocysts production was significantly higher in $100{\mu}M$ group than others (26.2% vs. 13.6%, 14.0%, 18.2%; P<0.05) respectively. The optimal concentrations of flavone significantly enhanced the percentages of ICM:TE than control group (43.8% vs. 37.6%; P<0.05) accompanied with significantly higher expression levels of reprogramming related genes. In conclusion, the optimal concentrations of $1{\mu}M$ during IVM and $100{\mu}M$ during IVC can significantly improve the production of porcine in-vitro embryos.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.301-306
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• 2004

This study was conducted to develop new markers at the region that was related to QTL affecting intramuscular fat and backfat thickness on chromosome 6q28 - 6q32 in pigs. Dozens of repeated sequences were founded using shotgun sequencing of several BAC clones corresponding to that region, of which five new microstellite markers that identified polymorphism were discovered. The mean number of alleles at each locus observed 2.13(KP0290F2), 4.63(KP0248Cll), 7.38(KP1231C91), 2.75(KPI23IC92) and 6.2S(KP1231C93) in 8 breeds(Landrace, Korean native pig, Duroc, Yorkshire, Berkshire, Wuzhishan pig, Xiang pig, Min pig). The average estimated heterozygosity values at each locus varied from 0.2100(KP0290F2) to 0.8304(KPI23IC91) in all populations. In other hand, the average allele of all loci WlL'I within range of 0.4517(Berkshire) and 0.6957 (Yorkshire). Of these markers, KP0248C11, KP1231C91 and KP1231C93 were identified to have optimal number of alleles, high heterozygosity values and low standard deviation values. Especially, KPI23IC91 and KPI231C93 might be considered as a useful marker for genetic mapping and diversity study.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.3
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• pp.439-445
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• 2017

Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• Environmental Analysis Health and Toxicology
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• v.15 no.4
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• pp.131-137
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• 2000

The release of hazardous waste materials into the environment poses serious risks in humans and ecosystems. The risk assessment of environmental pollutants including hazardous chemicals requires a comprehensive measurement of hazard and exposure of the chemicals that can be achieved by toxicity evaluation using a biological system such as biomarkers. In this report we have tried to develop a biomarker used to elucidate a molecular basis of, and to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. First, an attempt was made to clone tyrosine hydroxylase gene from Japanese medaka that would be a candidate for a biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon and their behavioral responses were observed . In this study we have successfully cloned a partial TH gene from the medaka fish through PCR screening of an ovary cDNA library. DNA sequencing analysis revealed that the amplified fragment was 327 bp encoding 109 amino acids. Comparing the DNA sequence of medaka TH with other species, TH gene revealed the DNA sequence was completely identical to that of rat TH. In the RT-PCR, 330 Up of mRNA was consistently amplified in all the treated samples including control There were no significant differences in the TH expression level regardless of treating concentrations (1∼5,000 ppb) and time (0∼48 hr) The reason appeared to be that RT-PCR was not performed using through a quantitative analysis normalized against an actin gene expression. Organ or tissue - specific detection of TH activity and mRNA as biomarkers will be a useful monitoring tool for neurobehavioral changes in fish influenced by toxic chemicals. Furthermore, quantitative analysis of locomotive patterns and its correlation with the neurochemical and molecular data would be highly useful in measuring toxicity and hazard ofvarious environmental pollutants.