• Journal of Embryo Transfer
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• v.21 no.1
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• pp.13-20
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• 2006

The purpose of this study was to develope the transgenic cattle expressing hFSH into the urine using the nuclear transfer. To produce the interest gene in urine, the specific vector was ligated with hFSH gene undo. maUII promoter. The fetal fibroblast cells (KbFF) were isolated from a 45-day male fetus. The hFSH gene was co-transfected with pcDNA3 (neo) vector to KbFF cells by electroporation. The gene-transfected cells were cultured with G-418 selection medium for 2 weeks. Selected colonies were confirmed by PCR. For nuclear transfer, enucleated bovine oocytes were transferred with hFSH transfected or nontransfected fetal fibroblasts. The cleavage and blastocyst formation rates were significantly lower (p<0.05) in cloned embryos transfected with hFSH gene (68.7% and 15.7%) than in those non-transfected (67.6% and 24.5 %), respectively. Apoptosis analysis showed no difference between hFSH transfected and non-transfected blastocysts (p>0.05). The blastocysts were transfected to 77 (control 24, hFSH 53) recipient cows. Two calves were born (1.9%) following transfer with NT embryos transfected with hFSH gene, but they were confirmed not to be transgenic calves. This result shows that the hFSH colonies were mixed with transfected and non transfected cells. Further research will be needed for selection and establishment of gene transfected cells.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.151-159
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• 2005

Injury caused by reactive oxygen species (ROS), known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Chloroplasts are specially sensitive to damage by ROS because electrons that escape from the photosynthetic electron transfer system are able to react with relatively high concentration of $O_2$ in chloroplasts. To cope with oxidative stress, plants have evolved an efficient ROS-scavenging enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), and low molecular weight antioxidants including ascorbate, glutathione and phenolic compounds. To maintain the productivity of plants under the stress condition, it is possible to fortify the antioxidative mechanisms in the chloroplasts by manipulating the antioxidation genes. A powerful gene expression system with an appropriate promoter is key requisite for excellent stress-tolerant plants. We developed a strong oxidative stress-inducible peroxidase (SWPA2) promoter from cultured cells of sweetpotato (Ipomoea batatas) as an industrial platform technology to develop transgenic plants with enhanced tolerance to environmental stress. Recently, in order to develop transgenic sweetpotato (tv. Yulmi) and potato (Solanum tuberosum L. cv. Atlantic and Superior) plants with enhanced tolerance to multiple stress, the genes of both CuZnSOD and APX were expressed in chloroplasts under the control of an SWPA2 promoter (referred to SSA plants). As expected, SSA sweetpotato and potato plants showed enhanced tolerance to methyl viologen-mediated oxidative stress. In addition, SSA plants showed enhanced tolerance to multiple stresses such as temperature stress, drought and sulphur dioxide. Our results strongly suggested that the rational manipulation of antioxidative mechanism in chloroplasts will be applicable to the development of all plant species with enhanced tolerance to multiple environmental stresses to contribute in solving the global food and environmental problems in the 21st century.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Molecules and Cells
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• v.28 no.1
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• pp.19-24
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• 2009

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and nonrecombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.298-305
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• 2016

Previously our laboratory showed that Pseudomonas alkylphenolica KL28 possesses two different lap and pcu gene clusters for p-cresol catabolism. In this study, additional gene cluster (pchACXF-pcaHG-orf4-pcaBC) has been identified to encode enzymes necessary for catabolism of p-cresol to ${\beta}$-carboxy-cis,cis-muconate. This gene cluster showed almost identical nucleotide sequence homologies to those in the plasmid of Pseudomonas putida NCIMB 9866 and 9869, British origins, indicating the possibility of a horizontal gene transfer. Through mutagenesis of each gene cluster and gfp-based promoter reporter assays, it has been shown that the three gene clusters are functionally operated and pch genes are induced by p-cresol. Furthermore, the pcu gene cluster of the three was shown to be dominantly expressed in utilization of p-cresol. Mutation of the pcu gene was defective in aerial structure formation under p-cresol vapor, indicating the utilization rate of carbon source is one of key elements for the multicellular development of this strain.

• Molecules and Cells
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• v.43 no.7
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• pp.645-661
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• 2020

Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20℃ but is inactivated more rapidly at 28℃. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.99-99
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• 2002

The hormone resistin is associated with typeII diabetes mellitus in rodent model. Resistin impairs glucose tolerance and insulin action. A new class of anti-diabetic drugs were called thiazolidinediones (TZDs) downregulates a resistin which is induced during adipocyte differentiation. But the connection between increased adiposity and resistin remains unknown. The objectives of this study was to clone a mouse resistin cDNA and to generate transgenic mice overexpressing mouse resistin gene. The 555 bp of mouse resistin was amplified from mob cDNAS by polymerase chain reaction (PCR) and cloned into pCR$\^$(R)/ 2.1 TOPO T-vector. Mouse resistin mRNA on the basis of Genbank sequence (acession no. AF323080). Then, the PCR product was cloned into pTargeT$\^$TM/ mammalian expression vector that has pCMV promoter and chimeric intron. Restriction enzyme analysis with BamH I and Not I was carried out to determine an orientation of the insert in the vector. The pCMV-mus/resistin gene was prepared from previous recombinant pTargeT$\^$TM/-mus/resistin by digestion of Bgl II, and has used for microinjection into pronuclei of one cell embryos. The microinjected embryos were transfered to pseudopregnant foster-mother. Mouse resistin expression was detected in transgenic F1 mice by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). Resistin gene expression mouse has heavier body weight which was measured higher level of plasma glucose than that of normal mouse. And in diet-induced experiments, the abdominal fat pads were isolated from each 24h starvation and re-feeding after fasting group mice that were assessed by RT-PCR analysis. In fasting group mice, resistin expression was higher than that of re-feeding group mice. This result suggests that the resistin gene overexpressing mice may be became to obesity and be useful as an animal disease model to be diabetes mellitus caused by insulin resistance of resistin.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.228-228
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• 2004

Interferon-tau (IFN-τ) is the primary agent responsible for maternal recognition of pregnancy in cattle. Bovine embryos begine to express IFN-τ as the blastocyst forms. Pregnancy recognition in ruminants occurs when IFN-τ from the trophoblast prevents the increase of oxytocin receptors, disrupting luteolytic pulses of prostaglandin (PG) F2a by oxytocin. The expression of IFN-τ is strongly associated with the degree of methylation of the CpG islands in promoter region. (omitted)

• Journal of Environmental Health Sciences
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• v.35 no.5
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• pp.343-354
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• 2009

Since Barker found associations between low birth weight and several chronic diseases later in life, the hypothesis of fetal origins of adult disease (aka, Barker Hypothesis) and epigenetics have been emerging as a new paradigm for geneenvironment interaction of chronic disease. Epigenetics is the study of heritable changes in gene silencing that occur without any change in DNA sequence. Gene expression can be regulated by several epigenetic mechanisms, including DNA methylation and histone modifications, which may be associated with chronic conditions, such as cancers, cardiovascular disease, and type-2 diabetes. One carbon metabolism which involves the transfer of a methyl group catalyzed by DNA methyltransferase is an important mechanism by which DNA methylation occurs in promoter regions and/or repetitive elements of the genome. Environmental factors may induce epigenetic modification through production of reactive oxygen species, alteration of methyltransferase activity, and/or interference with methyl donors. In this review, we introduce recent studies of epigenetic modification and environmental factors, such as heavy metals, environmental hormones, air pollution, diet and psychosocial stress. We also discuss epigenetic perspectives of early life environmental exposure and late life disease occurrence.