• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.9
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• pp.1226-1234
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• 2012

3,4,3',5'-Tetrahydroxy-trans-stilbene (piceatannol) is a derivative of resveratrol with a variety of biological activities, including anti-inflammatory, anti-proliferative, and anti-cancer activities. We assessed the mechanisms by which piceatannol inhibits inflammatory responses using lipopolysaccharide (LPS)-treated Raw264.7 murine macrophages. Piceatannol (0~10 ${\mu}mol/L$) decreased LPS-induced release of nitric oxide, tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-6, IL-$1{\beta}$, and inhibited LPS-induced protein expression of inducible nitric oxide synthase (iNOS). Activation of nuclear factor-kappaB (NF-${\kappa}B$), activator protein (AP)-1, and signal transducer and activator of transcription 3 (STAT3) are crucial steps during an inflammatory response. Piceatannol prevented LPS-induced degradation of inhibitor of ${\kappa}B$ ($I{\kappa}B$), translocation of p65 to the nucleus, and phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Additionally, piceatannol inhibited LPS-induced phosphorylation of STAT3 and IL-6-induced translocation of STAT3 to the nucleus. Furthermore, piceatannol increased the protein and mRNA levels of hemeoxygenase (HO)-1, the rate-limiting enzyme of heme catabolism that plays a critical role in mediating antioxidant and anti-inflammatory effects. Piceatannol further induced antioxidant response elements (ARE)-driven luciferase activity in Raw264.7 cells transfected with an ARE-luciferase reporter construct containing the enhancer 2 and minimal promoter region of HO-1. These results suggest that piceatannol exerts anti-inflammatory effects via the down-regulation of iNOS expression and up-regulation of HO-1 expression.

• Proceedings of the Korean Society of Toxicology Conference
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• 2000.11a
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• pp.45-67
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• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Clinical and Experimental Pediatrics
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• v.49 no.12
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• pp.1340-1347
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• 2006

Purpose : The aim of the present study was to investigate the role of polymorphic cytosine adenine (CA) repeat of the IGF-I gene in the age-related alterations of serum IGF-I levels in healthy children. Methods : Two hundred and forty three normal healthy children (136 boys; 107 girls) aged between 7 and 15 years were enrolled in the present study. The primers were designed to cover the promoter regions containing the polymorphic CA repeat. Data were analyzed using GeneMapper software, version 3.7. All analyses were performed using MEDCALC software packages. Results: Deletion of 2 bp (G, A) following 3' of CA repeat were observed in all Korean children. The CA repeat sequences ranged from 17 to 23, and 19 CA repeat were the most common with an alleles frequency of 39.3 percent. Considering genotypes, 63.8 percent of subjects were homozygote or heterozygote for 19 CA repeat (192 bp allele), suggesting that this is wild type allele from which all other alleles originated in Korean children. Homozygote for 19 CA repeat were 14.7 percent, heterozygote for 19 CA repeat was 49.1 percent and 19 CA noncarriers totalled 36.2 percent. In 19 CA repeat noncarriers, the mean height, weight and serum IGF-I level were lower compared with those of 19 CA homozygous carriers, but statistically not significant. Correlations between serum IGF-I level and age according to the IGF-I genotypes revealed statistically significant relationships in the all groups, in the 19 CA repeat carrier group and, even in the noncarrier group. Conclusions : There were no significant differences of the mean height, weight and serum IGF-I levels among three different genotype groups. Also, there were no significantly different correlations between 19 CA repeat polymorphisms and serum IGF-I levels, according to genotype. Our results suggest that the IGF-I 19 CA repeat gene polymorphism is not associated with circulating IGF-I levels in healthy children.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.251-257
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• 2011

Rice is the most important crop as a model plant for functional genomics of monocotyledons. Rice is usually transformed using Agrobacterium tumefaciens. However, the transformation efficiency using previous method is still low. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the inoculation and co-cultivation step. We directly inoculated Agrobacterium containing a CIPK15 gene under the control of CaMV 35S promoter and NOS terminator in the pCAM1300 vector into the pre-soaked seeds in N6D media for 24 hours. After 7 days of culture at $25^{\circ}C$, calli were formed on seeds cultured on the co-cultivation medium containing an antioxidant compound (1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (3 mg/L silver nitrate). We obtained 35 and 22 transgenic plants in rice cultivars, Gopumbyeo and Ilpumbyeo, with increase of transformation efficiency by 30.4% and 22.6%, respectively compared to the general transformation method. The new method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.5 no.1
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• pp.48-56
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• 2005

Purpose: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000~15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl-CpG-binding protein). In this study, we carried out diagnostic mutational analysis of MECP2 gene in RTT patients. Methods: We analyzed four exons and putative promoter of MECP2 gene from the peripheral blood of 43 Korean patients with RTT by PCR-RFLP and direct sequencing. Results: Mutations were detected in MECP2 gene about 60.5% of patients. The mutations consisted of 14 different types including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, P385fsX409) were newly identified and these were determined as disease-causing mutations by PCR-RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified with high frequency. An intronic SNP (IVS3+23C>G) was newly identified in only three of the patients. It may be a disease-related and Korea-specific SNP with RTT. The L100V and A201V have been reported to be unclassified variant and SNP. However, these mutations were not found in more than 100 normal Korean control samples. These base substitutions seem to be the disease-causing mutations in Korean RTT contrary to previous studies. Conclusion: Disease-causing mutations and polymorphisms would be very important for diagnosing of RTT in Korean. The experimental procedure used in this study might be considered for molecular biologic diagnosis used in clinical field.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.10a
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• pp.14-14
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• 2010

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed two different kinds of transgenic potato plants (Solanumtuberosum L. cv. Taedong Valley) overexpressing strawberry GalUR and mouse GLoase gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the these genes in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid(AsA) levels in transgenic tubers were determined by high-performance liquid chromatography(HPLC). The over-expression of these genes resulted in 2-4 folds increase in AsA intransgenic potato and the levels of AsA were positively correlated with increased geneactivity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen(MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of these gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. We have also investigated the mechanism of the abiotic stress tolerance upon enhancing the level of the ascorbate in transgenic potato. The transgenic potato plants overexpressing GalUR gene with enhanced accumulation of ascorbate were investigated to analyze the antioxidants activity of enzymes involved in the ascorbate-glutathione cycle and their tolerance mechanism against different abiotic stresses under invitro conditions. Transformed potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed significant increase in the activities of superoxide dismutase(SOD, EC 1.15.1.1), catalase, enzymes of ascorbate-glutathione cycle enzymes such as ascorbate peroxidase(APX, EC 1.11.1.11), dehydroascorbate reductase(DHAR, EC 1.8.5.1), and glutathione reductase(GR, EC 1.8.1.7) as well as the levels of ascorbate, GSH and proline when compared to the untransformed tubers. The increased enzyme activities correlated with their mRNA transcript accumulation in the stressed transgenic tubers. Pronounced differences in redox status were also observed in stressed transgenic potato tubers that showed more tolerance to abiotic stresses when compared to untransformed tubers. From the present study, it is evident that improved to lerance against abiotic stresses in transgenic tubers is due to the increased activity of enzymes involved in the antioxidant system together with enhanced ascorbate accumulated in transformed tubers when compared to untransformed tubers. At moment we also investigating the role of enhanced reduced glutathione level for the maintenance of the methylglyoxal level as it is evident that methylglyoxal is a potent cytotoxic compound produced under the abiotic stress and the maintenance of the methylglyoxal level is important to survive the plant under stress conditions.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.