[Show abstract][Hide abstract] ABSTRACT: Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409].
[Show abstract][Hide abstract] ABSTRACT: Tracheal epithelial cells (TECs) are an important tool for studies of viral respiratory diseases. Primary TECs have been cultured from human, mouse and hamster. It is also necessary to diagnose viral respiratory disease and reveal infection mechanisms in chicken. In this study, we isolated tracheal epithelial layers from tracheal of 20-day-old chicks and cultured primary TECs from the isolated layers. Ciliated cells which were a typical morphology of TECs were observed in cultured primary TECs and maintained until cell passage 5 (15 to 20 days). When we analyzed expression patterns of epithelial marker genes (retinoic acid responder, FGF-binding protein, virus activating protease (VAP) in TECs compared to immortalized chicken embryonic fibroblast cell line (DF-1), all the marker genes are highly expressed in TECs than in DF-1. When TECs were cultured with 0.1 and 1 MOI of ND virus (rNDV-GFP strain) to test the susceptibility of TECs for ND virus, 12.6% and 48.2% of the incubated TECs were infected respectively. In addition, when DF-1 was incubated with 1 MOI of ND virus, the virus infection rate of DF-1 was three times lower than the virus infection rate of TECs. These data could contribute to study infection mechanisms of viral respiratory diseases and control them in chicken.
Korean Journal of Poultry Science. 01/2013; 40(4).
[Show abstract][Hide abstract] ABSTRACT: Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.
[Show abstract][Hide abstract] ABSTRACT: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.
Biochemical and Biophysical Research Communications 07/2012; 424(4):765-70. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza A virus infection is a great threat to avian species and humans. Targeting viral proteins by antibody has a limited success due to the antigen drift and shift. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a sequence-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)-encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18-nucleotide single stranded nucleic acid substrate, dubbed asNP(18), the sequence of which is unique to the NP-vRNA. We isolated a 3D8 VL variant, NP25, that had ∼15-fold higher affinity (∼54nM) and ∼3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP(18)-selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any significant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP(18) sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a new strategy of targeting viral genomic RNA for degradation by antibody for the prevention of influenza virus infection in humans and animals.
Antiviral research 03/2012; 94(2):157-67. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to examine the effect of different sera and serum-like substances on the preimplantation development of porcine parthenogenetic embryos. Chemically activated (calcium ionophore A23187+cytochalasin B) pig oocytes were pre-cultured for five days. On day 5, the parthenogenetic embryos were treated with porcine follicular fluid (PFF), fetal bovine serum (FBS), horse serum (HS) or porcine serum albumin (PSA), and were cultured two more days. Horse serum was found to be the most effective protein source in enhancing parthenote development judging by blastocyst formation and hatching. Next, three different concentrations of HS (10, 20 and 30%) were used to determine the optima HS concentration needed to improve the development of porcine parthenogenetic embryos. All HS concentrations increased the blastocyst cell number and decreased the incidence of blastocyst apoptotic cells with 20% being the most effective. In conclusion, horse serum enhanced parthenogenetic embryo development and the quality of porcine parthenogenetic embryos.
[Show abstract][Hide abstract] ABSTRACT: Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens.
Reproduction Fertility and Development 12/2011; 24(1):220-1. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.
[Show abstract][Hide abstract] ABSTRACT: In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.
Bioscience Biotechnology and Biochemistry 05/2011; 75(4):646-9. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.
Biochemical and Biophysical Research Communications 08/2009; 387(2):266-71. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ectopic expression of the structural protein Pr55(Gag) of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5'-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3'-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55(Gag) in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55(Gag) derived from this JEV replicon RNA appeared to be increased by approximately 3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55(Gag) proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55(Gag) could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55(Gag) by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.
Virus Research 06/2009; 144(1-2):298-305. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the expression of the mouse complement component C1rA (mC1rA) in IFN-gamma-stimulated mouse keratinocytes (Pam 212) and found that it was upregulated. To analyze the mechanism involved, we cloned the 2,150 bp 5'-flanking region of mC1rA by the vectorette-PCR technique, and identified the transcription start site of mC1rA by rapid amplification of complementary DNA ends. Analysis of the 5' sequence revealed putative binding sites for activator protein 1, CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 1 (STAT-1), IFN-regulatory factor-1 (IRF-1), and others. We detected transcriptional activation dependent on this upstream region in reporter gene assays and Northern blots. To identify the cis-acting regulatory elements involved, we analyzed serial deletion constructs of the promoter using luciferase reporters. The -80 to -19 bp region, which contains a putative IRF-1 binding site, was required for both basal promoter activity and responses to IFN-gamma. The use of site-directed point mutations, electrophoresis mobility shift assays, and supershift assays indicated that the putative IRF-1 binding site was essential for both IFN-gamma-dependent and -independent transcriptional activity of the mC1rA promoter. We conclude that IFN-gamma stimulates mC1rA gene expression via IRF-1 in mouse keratinocytes.
Journal of Investigative Dermatology 06/2007; 127(5):1187-96. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin during the laying period. In this study, we identified oviduct-specific proteins in hens during the egg-laying period by proteomic analysis. Proteins extracted from the magnum of hens of different ages (5, 35, and 65 weeks) were analyzed by two-dimensional gel electrophoresis to compare the intensity of proteins among samples. Approximately 300 spots were detected on each gel. Based on the comparison of image gels, we found that the intensity of eight spots in 35-week magnums was increased at least by 2-fold compared with the others. Five of the eight spots were identified as calumenin, acidic ribosomal phosphoproteins (ARP), prohibitin, heart fatty acid-binding protein, and anterior gradient-2 (AGR-2). In particular, ARP and AGR-2 were highly expressed in 35- week magnums compared with 5- and 65-week magnums. In addition, the level of these proteins was consistent with their RNA levels. Expression of AGR-2 mRNA was detected in the mature magnum, whereas no signal was observed in premature tissue. Among various tissues, expression of AGR-2 mRNA was highest in the magnum, high in the isthmus, and five fold lower in muscle. It was undetectable in the liver and in other tissues (heart and kidney). However, the mRNA levels of other proteins were ubiquitous among tissues. In transcriptional activity of AGR-2, a 3.0 kb fragment of promoter region containing potential estrogen receptor binding sites had enhanced its activity strongly. In conclusion, these results suggest that AGR-2 has functional regulatory roles in the chicken oviduct during the egglaying period.
Journal of biochemistry and molecular biology 03/2007; 40(2):212-7. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report four variants and alternative promoter usage for the mouse acyl-CoA synthetase 6 (mAcsl6) gene. The variants, which were organized into 26 exons and 25 introns spanning 55 kb of DNA on mouse chromosome 11, were classified according to their 5'-UTRs and alternative splicing of exon 13. Alignment of the nucleotide sequences showed that the mAcsl6 variant 1 (mAcsl6_v1) and mAcsl6_v2 used a different promoter and had different splicing patterns than mAcsl6_v3 and mAcsl6_v4. The results of the promoter analysis suggest that the mAcsl6 promoter 1 (mAcsl6_pr1) region has a negative regulatory function. To verify this result, we constructed id vector constructs that contained the promoter regions mAcsl6_pr1 and 2, and the chimeric transcript. Although the mAcsl6_pr1 region was deleted, the mAcsl6_v1 and 2 transcripts were detected consistently.
Biochemical and Biophysical Research Communications 03/2005; 327(1):84-93. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-kappaB and AP-1, and the IL-8 promoter activity. However, cotransfection with IkappaB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-kappaB pathway.
Biochemical and Biophysical Research Communications 05/2004; 317(1):276-84. · 2.28 Impact Factor