[show abstract][hide abstract] ABSTRACT: We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.
[show abstract][hide abstract] ABSTRACT: Tissue engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic Bcl-2 gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function.
Tissue Engineering Part A 10/2012; · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: One of the major goals of gene therapy is to regulate the expression of therapeutic genes in desired cells or tissues. For this purpose, heat-inducible vectors have been exploited for cancer gene therapy combined with hyperthermia, which can result in considerable improvement of therapeutic effects. In the present study, we constructed a novel heat-inducible gene expression system incorporating a transactivation system with a positive feedback loop of transcriptional amplification. The target gene expression mediated by the transactivator under the control of a heat shock protein 70B' promoter is enhanced by self-promoted transactivator gene expression. This expression system showed tight control of target gene expression together with high-level expression; enhanced expression of the reporter gene was observed in transfected cells upon heat treatment, while negligible gene expression was detected in non-heated cells. When a therapeutic gene was used as the target gene, a considerable cytotoxic effect was observed after heat treatment of cancer cells transfected with the plasmids. The heat-induced transgene expression system is a promising new approach for the development of both a safe and effective vector for hyperthermia-based cancer gene therapy.
Journal of Bioscience and Bioengineering 05/2012; 114(4):460-5. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Patients with obstructive sleep apnea syndrome (OSAS) are always exposed to intermittent hypoxia and reoxygenation. The metabolic syndrome (MetS) and OSAS are also known to accelerate atherosclerosis, diabetes, and dyslipidemia. Therefore, nasal continuous positive airway pressure (CPAP) therapy may have beneficial effects in patients with the MetS and OSAS.
This study in patients with the MetS and OSAS tested the validity of the hypothesis that chronic CPAP therapy improves factors involved in atherosclerosis, including impaired endothelial function.
Thirty-two patients (19 males and 13 females, mean age 54 ± 9 y) diagnosed with the MetS and OSAS were enrolled in the study and received CPAP therapy for 3 months. Vascular function was investigated by measuring forearm blood flow (FBF) responses to reactive hyperemia (RH) using venous occlusion strain-gauge plethysmography. Biochemical markers were also measured before and after this procedure.
Basal apnea-hypopnea index was statistically correlated with FBF response to RH. The FBF response to RH was increased significantly after 3 months of CPAP therapy. A significant increase in plasma nitric oxide levels and a decrease in the levels of asymmetrical dimethylarginine, thiobarbituric acid reactive substance, soluble Fas ligand, and soluble CD40 ligand were detected after CPAP therapy. The plasma concentrations of tumor necrosis factor-α, interleukin (IL)-6, and IL-8 also decreased significantly with CPAP therapy, whereas IL-1β levels remained unchanged.
Continuous positive airway pressure therapy has beneficial effects on vascular function and inflammatory and oxidative stress in patients with the MetS and OSAS.
[show abstract][hide abstract] ABSTRACT: Purpose: Control of therapeutic gene expression in tumours is a major goal of gene therapy research, as it can restrict cytotoxic gene expression in cancer cells. In addition, the combination of hyperthermia with gene therapy through the application of heat-inducible vectors can result in considerable improvements in therapeutic efficiency. In this study, to combine heat-inducibility with high-level transgene expression, we developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator. Materials and methods: A hybrid promoter was generated by placing the heat shock protein (HSP) 70B' promoter under the tetracycline-repressor responsive element sequence, and a reporter/therapeutic gene expression plasmid was constructed by placing a reporter/therapeutic gene under the control of this hybrid promoter. Results: When the transactivator expression plasmid harbouring an expression cassette of the tetracycline-responsive transactivator gene was co-transfected with a reporter gene expression plasmid, the reporter gene expression was controlled by heat treatment. With this system, high levels of heat-induced transgene expression were observed compared to that from the HSP promoter alone without the transactivator. Evaluation of in vitro therapeutic effects using cancer cell lines revealed that therapeutic gene expression effectively caused cell death in a greater percentage of the cells. Conclusion: These findings indicate that this strategy improves the efficacy of cancer gene therapy.
International Journal of Hyperthermia 01/2012; 28(8):788-98. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.
Journal of Bioscience and Bioengineering 11/2011; 113(3):381-8. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Embryoid bodies resemble post-implantation egg-cylinder stage embryos and are used to differentiate embryonic stem cells in vitro. In this study, we enriched mouse vasa homolog-positive germ cells from embryoid bodies after 8d of differentiation using a magnetic separation method with magnetite cationic liposomes.
Journal of Bioscience and Bioengineering 05/2011; 112(2):184-7. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chinese hamster ovary (CHO) cell lines for the production of recombinant human erythropoietin (hEpo) and erythropoietin/Fc
fusion protein (hEpo/Fc) were established. Productivities of the proteins by transient gene expression were up-regulated by
the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) residing downstream hEpo and hEpo/Fc sequences.
The cells have shown to correctly synthesize bioactive hEpo and hEpo/Fc proteins. Despite that hEpo presented a higher activity
in vitro, hEpo/Fc bioactivity was partially conserved.
KeywordsRecombinant human erythropoietin (hEpo) and hEpo/fc fusion protein-Chinese hamster ovary (CHO) cells-Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)
[show abstract][hide abstract] ABSTRACT: To present a new method called magnetolipofection which can transfect cells in a specific area of the retinal pigment epithelium (RPE) by magnetic force as a non-viral gene transfection.
ARPE-19 (a human RPE cell line) cells were cultured with a mixture of cationic lipid, plasmid DNAs and magnetite nanoparticles. A sheet of ARPE-19 cells was transfected in the vertical direction by placing a magnet under the centre of the culture plate. Horizontal gene transfection was also performed.
When magnetolipofection was performed in the vertical direction, there was a significantly larger number of green fluorescent protein (GFP)-positive cells where the magnet was placed than in the peripheral area, and the number was equivalent to the number transfected with Lipofectamine2000. In the horizontal direction, there was also a significantly larger number of GFP-positive cells, but there was almost no gene transfer detected using Lipofectamine2000.
The area of gene transfection can be controlled by the placement of a magnet in the area selected to be transfected in vitro by magnetolipofection. This method can be used to transfect RPE cells in selected areas which should be helpful for experimental and clinical applications.
The British journal of ophthalmology 12/2009; 94(8):1074-7. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: An epithelial cell adhesion molecule, E-cadherin was expressed in NIH3T3 fibroblasts, and cell-cell interactions between keratinocytes and fibroblasts, or between hepatocytes and fibroblasts were artificially engineered. When the E-cadherin-expressing NIH3T3 cells were co-cultured with rat hepatocytes, the cell-cell contacts were formed with high frequency, and enhanced albumin secretion was observed.
Journal of Bioscience and Bioengineering 06/2008; 105(6):679-82. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, we applied an ultra-water-repellent film to cell culture. We cultured cells in droplets on the film and fabricated cell aggregates. Furthermore, we allocated cells on micropatterned surfaces consisting of ultra-water-repellent regions and cell culture-treated regions. The results show that the material is useful for cell culture.
Journal of Bioscience and Bioengineering 12/2007; 104(5):420-3. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small bowel bacterial overgrowth (SBBO) may be associated with malnutrition, diarrhea, and weight loss. Recently, bone mineral density (BMD) in patients with SBBO was reported to be lower, and SBBO may be an important factor in the development of metabolic bone disease. However, the subjects in these studies were relatively young patients with intestinal diseases. There is no information on the effect of SBBO on BMD in older people.
Seventeen relatively active and 33 disabled older people participated in this study. SBBO was determined by a breath hydrogen (H2) test after ingestion of a glucose solution. BMD of the lumbar spine and femur were measured using a dual energy X-ray absorptiometry scan (DEXA).
One healthy control and 11 disabled subjects were SBBO-positive. The Z-scores of the lumbar spine were not statistically different between groups, and a high incidence of disorders, >70%, was seen in all groups. On the other hand, there were significant differences in the femoral BMD between the healthy controls and the SBBO-negative (P<0.001) and SBBO-positive (P<0.05) groups. No significant difference was seen in femoral BMD between SBBO-positive and SBBO-negative institutionalized people.
SBBO seems to have little effect on BMD in people approximately 80 years old.