九州大学-研究者情報 [河邉佳典 (准教授) 工学研究院化学工学部門]

河邉佳典（かわべよしのり）

データ更新日：2024.04.03

准教授　／　工学研究院化学工学部門分子・生物システム工学講座

原著論文

1.	Md. Rashidur Rahman, Yoshinori Kawabe, Kozumi Suzuki, Satoshi Chen, Yuki Amamoto, Masamichi Kamihira, Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen-binding domain, Biotechnology Journal, doi: 10.1002/biot.202300362, 19, 1, e2300362, 2024.01, [URL].
2.	Feiyang Zheng, Yoshinori Kawabe, Masamichi Kamihira, RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells, ACS Synthetic Biology, https://doi.org/10.1021/acssynbio.3c00472, 13, 1, 230-241, 2024.01, [URL].
3.	Binbin Ying, Yoshinori Kawabe, Feiyang Zheng, Yuki Amamoto, Masamichi Kamihira, High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells, Cells, https://doi.org/10.3390/cells12222638, 12, 22, 2638, 2023.11, [URL].
4.	Yusuke Sakai, Yoshihiro Kubo, Nana Shirakigawa, Yoshinori Kawabe, Masamichi Kamihira, Hiroyuki Ijima, Promotion of Cyst Formation from a Renal Stem Cell Line Using Organ-Specific Extracellular Matrix Gel Format Culture System, Gels, 10.3390/gels8050312, 8, 5, 312-312, 2022.05, [URL].
5.	Masanobu Horie, Noriko Yamano-Adachi, Yoshinori Kawabe, Hidenori Kaneoka, Hideaki Fujita, Eiji Nagamori, Ryosuke Iwai, Yasushi Sato, Kei Kanie, Seiichi Ohta, Masaharu Somiya, Kosuke Ino, Recent advances in animal cell technologies for industrial and medical applications, Journal of Bioscience and Bioengineering, https://doi.org/10.1016/j.jbiosc.2022.03.005, 133, 6, 509-514, 2022.04, [URL].
6.	Hiroyuki Kitano, Yoshinori Kawabe, Masamichi Kamihira, HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions, Cells, https://doi.org/10.3390/cells11071194, 11, 7, 1194, 2022.03, [URL].
7.	Yoshinori Kawabe, Masamichi Kamihira, Novel cell lines derived from Chinese hamster kidney tissue, PLoS One, doi: 10.1371/journal.pone.0266061., 17, 3, e0266061, 2022.03, [URL].
8.	Kantaro Yoshioka, Akira Ito, Masanobu Horie, Kazushi Ikeda, Sho Kataoka, Keiichiro Sato, Taichi Yoshigai, Hidetoshi Sakurai, Akitsu Hotta, Yoshinori Kawabe, Masamichi Kamihira, Contractile activity of myotubes derived from human induced pluripotent stem cells: A model of duchenne muscular dystrophy, Cells, https://doi.org/10.3390/cells10102556, 10, 10, 2556, 2021.10, [URL].
9.	Shinya Masumoto, Akihiko Ono, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Hypoxia-responsive expression of vascular endothelial growth factor for induction of angiogenesis in artificial three-dimensional tissues, J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2021.06.010, 132, 4, 399-407, 2021.10, [URL].
10.	Guan Huang$, Yoshinori Kawabe$, Kazuki Shirakawa, Tatsuki Akiyama, Masamichi Kamihira ($Co-1st author), Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system  , J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2021.07.006, 132, 5, 469-478, 2021.07, [URL].
11.	Feiyang Zheng, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Kyoka Nishihata, Souichiro Yoshida, Akira Ito, Masamichi Kamihira, LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells, Biotechnol J, https://doi.org/10.1002/biot.202000620, 16, 7, e2000620, 2021.07, [URL].
12.	Kantaro Yoshioka, Akira Ito, Md Arifuzzaman, Taichi Yoshigai, Fangming Fan, Kei-Ichiro Sato, Kazunori Shimizu, Yoshinori Kawabe, Masamichi Kamihira, Miniaturized skeletal muscle tissue fabrication for measuring contractile activity, J Biosci Bioeng, 10.1016/j.jbiosc.2020.11.014, 131, 4, 434-441, 2021.04, [URL].
13.	Hiroyuki Kitano, Yuki Nagae, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Development of a genetically modified hepatoma cell line with heat-inducible high liver function, Cytotechnology, https://doi.org/10.1007/s10616-021-00457-4, 73, 3, 353-362, 2021.06, [URL].
14.	Ryusei Iwao, Yoshinori Kawabe, Mai Murakami, Akira Ito, Masamichi Kamihira, Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307001, 333, 07001, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01, [URL].
15.	Feiyang Zheng, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Shoichiro Yoshida, Akira Ito, Masamichi Kamihira, Retrotransposon-mediated Gene Transfer for Animal Cells, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307002, 333, 07002, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01, [URL].
16.	Kazuki Shirakawa, Yoshinori Kawabe, Guan Huang, Akira Ito, Masamichi Kamihira, Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxP Recombination System, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307003, 333, 07003, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01, [URL].
17.	Shinya Masumoto, Akira Ito, Akihiko Ono, Tomonaga Sato, Masaki Yamaguchi, Yoshinori Kawabe, Masamichi Kamihira, Construction of Hypoxia-Responsive VEGF Gene-Expression System Using Synthetic Biological Approach, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307005, 333, 07005, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01, [URL].
18.	Hiroyuki Kitano, Manuel Souvervielle Soto, Yuto Sonoda, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307007, 333, 07007, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01, [URL].
19.	Kantaro Yoshioka, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira., Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons, J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2019.10.004, 129, 4, 486-493, 2020.04, [URL].
20.	Ming Shi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2019.09.011, 129, 3, 363-370, 2020.03, [URL].
21.	Akira Ito, Ryoji Teranishi, Kazuki Kamei, Masaki Yamaguchi, Akihiko Ono, Shinya Masumoto, Yuto Sonoda, Masanobu Horie, Kawabe Yoshinori, Masamichi Kamihira, Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2019.03.008, 128, 3, 355-364, 2019.09, [URL].
22.	Paerhati P, Ito A, Yoshioka K, Iwamoto K, Fujiwara S, Horie M, Kawabe Y, Kamihira M., Neural differentiation of mouse induced pluripotent stem cells using cadherin gene-engineered PA6 feeder cells., J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2018.10.009, 127, 5, 633-640, 2018.11, [URL].
23.	Arifuzzaman M, Ito A, Ikeda K, Kawabe Y, Kamihira M., Fabricating Muscle-Neuron Constructs with Improved Contractile Force Generation, Tissue Eng Part A, http://doi.org/10.1089/ten.tea.2018.0165, 25, 7-8, 563-574, 2018.10, [URL].
24.	Yoshinori Kawabe, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05, [URL].
25.	Yoshinori Kawabe, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Development of retroviral vectors capable of site-specific gene insertion together with protein delivery, BMC Proceedings, https://doi.org/10.1186/s12919-018-0097-x, 2018, 12(Suppl 1):P-327, 2018.03, [URL].
26.	Xue Wang, Yoshinori Kawabe, Takeshi Hada, Akira Ito, Masamichi Kamihira, Cre-mediated transgene integration in Chinese hamster ovary cells using minicircle DNA vectors, Biotechnology Journal, 10.1002/biot.201800063, 13, 7, e1800063, 2018.07, [URL].
27.	Hideaki Yamamoto, Jane Marie Tonello, Takanori Sambuichi, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.07.011, 125, 1, 131-139, 2018.01, [URL], New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research..
28.	Jane Marie Tonello, Saori Kawashima, Kazuki Sato, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Three-dimensional culture of a genetically modified hepatoma cell line using macroporous gelatin beads, Cytotechnology, 10.1007/s10616-017-0117-0, 69, 6, 925-931, 2017.12, [URL], Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research..
29.	Xue Wang, Kawabe Yoshinori, Risa Kato, Takeshi Hada, Akira Ito, Yoshimasa Yamana, Masako Kondo, Masamichi Kamihira, Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.05.017, 124, 5, 583-590, 2017.11, [URL].
30.	Akihiko Ono, Akira Ito, Tomonaga Sato, Masaki Yamaguchi, Taiga Suzuki, Yoshinori Kawabe, Masamichi Kamihira, Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain., J Biosci Bioeng, 10.1016/j.jbiosc.2017.02.012, 124, 1, 115-124, 2017.07.
31.	Kazushi Ikeda, Akira Ito, Masanori Sato, Shota Kanno, Yoshinori Kawabe, Masamichi Kamihira, Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs., J Tissue Eng Regen Med., 10.1002/term.2030, 11, 1322-1331, 2017.05.
32.	Kazushi Ikeda, Akira Ito, Ryusuke Imada, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model, Sci Rep, 10.1038/srep44570, 7, 44570, 2017.03.
33.	Momoko Kitaoka, Ayaka Naritomi, Yoshinori Kawabe, Masamichi Kamihira, Noriho Kamiya, Masahiro Goto, Transcutaneous pollinosis immunotherapy using a solid-in-oil nanodispersion system carrying T cell epitope peptide and R848, Bioeng Transl Med , doi: 10.1002/btm2.10048, 2, 1, 102-108, 2017.02.
34.	Kawabe Yoshinori, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira, Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2016.10.011, 123, 3, 390-397, 2017.03, [URL].
35.	Masanobu Horie, Anuj Tripathi, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Magnetic nanoparticles Functionalization and manufacturing of pluripotent stem cells, Advanced Structured Materials, 10.1007/978-981-10-3328-5_9, 66, 363-383, 2017.01, [URL], Regenerative medicine uses cell alone or in combination with carrier to deliver at the required site for restoring the normal functions of diseased or degenerated tissue. Various strategies to restore tissue functions involve specific cell types, scaffolds and delivery processes that are still in developmental stage. Obtaining sufficient quantity of cells by non-invasive approach for the application in regenerative medicine is still a challenge. Pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), possess the inherent ability of self-renewal and differentiation into many cell types. In particular, iPSCs are of a special interest because patient-derived iPSCs have the ability to reproduce patient-specific clinical conditions. The development of manufacturing systems for PSCs, including cell culture engineering, is a challenging research field for the clinical application of PSCs such as in regenerative medicine. One of these manufacturing systems uses magnetic nanoparticles which are well known for their application in magnetic resonance imaging and magnetic hyperthermia. Besides, this chapter is focused on the basics of magnetic nanoparticles, its functionalization and further applications of a magnetic force-based cell manufacturing system for pluripotent stem cells. Indeed, we have developed a procedure in which cells are labeled with magnetite cationic liposomes via electrostatic interaction between the positively charged liposomes and the target cells. The culture system may provide a useful tool for studying the behavior of PSCs and an efficient way of PSCs manufacturing for clinical applications..
36.	Kawabe Yoshinori, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors, Biotechnology and Bioengineering, 10.1002/bit.25923, 113, 7, 1600-1610, 2016.07, [URL].
37.	Kazushi Ikeda, Akira Ito, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Improved contractile force generation of tissue-engineered skeletal muscle constructs by IGF-I and Bcl-2 gene transfer with electrical pulse stimulation, Regenerative Therapy, 10.1016/j.reth.2015.12.004, 3, 38-44, 2016.03, [URL], Introduction: Tissue-engineered skeletal muscle constructs should be designed to generate contractile force with directional movement. Because electrical impulses from a somatic nervous system are crucial for in vivo skeletal muscle development, electrical pulse stimulation (EPS) culture as an artificial exercise is essential to fabricate functional skeletal muscle tissues in vitro. To further improve muscle functions, the activation of cell-signaling pathways from myogenic growth factors, such as insulin-like growth factor (IGF)-I, is also important. Because tissue-engineered skeletal muscle constructs should maintain a high cell-dense structure, the expression of an anti-apoptotic factor, such as B-cell lymphoma 2 (Bcl-2), could be effective in preventing cell death. Methods: In the present study, myoblasts were genetically modified with inducible expression units of IGF-I and Bcl-2 genes, and the tissue-engineered skeletal muscle constructs fabricated from the myoblasts were cultured under continuous EPS. Results: Overexpression of IGF-I gene induced muscular hypertrophy in the muscle tissue constructs, and Bcl-2-overexpressing myoblasts formed significantly cell-dense and viable muscle tissue constructs. Furthermore, the combination of IGF-I and Bcl-2 gene transfer with EPS culture highly improved the force generation of the tissue-engineered skeletal muscle constructs. Conclusions: This approach has the potential to yield functional skeletal muscle substitutes with high force generation ability..
38.	Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Akira Ito, Masamichi Kamihira, Cre-mediated cellular modification for establishing producer CHO cells of recombinant scFv-Fc, BMC Proceedings, 10.1186/1753-6561-9-S9-P5, 9(Suppl 9):P5, 2015.12, [URL].
39.	Momoko Kitaoka, Yoko Shin, Noriho Kamiya, Yoshinori Kawabe, Masamichi Kamihira, Masahiro Goto, Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology., AAPS PharmSciTech, 10.1208/s12249-015-0333-x, 16(6):1418-24, 2015.12.
40.	Akihiko Ono, Akira Ito, Taiga Suzuki, Masaki Yamaguchi, Yoshinori Kawabe, Masamichi Kamihira, DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification, J Biosci Bioeng. , doi: 10.1016/j.jbiosc.2015.02.009., 120(4):463-6, 2015.10.
41.	Tetsushi Sakuma, Mitsumasa Takenaga, Kawabe Yoshinori, Takahiro Nakamura, Masamichi Kamihira, Takashi Yamamoto, Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids, International Journal of Molecular Sciences, 10.3390/ijms161023849, 16, 10, 23849-23866, 2015.10, [URL], Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins..
42.	Takanori Inao, Kawabe Yoshinori, Takuro Yamashiro, Yujiro Kameyama, Xue Wang, Akira Ito, Masamichi Kamihira, Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.11.019, 120, 1, 99-106, 2015.07, [URL], Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an invitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS..
43.	Masanobu Horie, Akira Ito, Takeshi Maki, Yoshinori Kawabe, Masamichi Kamihira, Magnetically labeled feeder system for mouse pluripotent stem cell culture, J Biosci Bioeng, doi: 10.1016/j.jbiosc.2014.10.020. , 119, 5, 614-616, 2015.05.
44.	Akira Ito, Masahiro Yamamoto, Kazushi Ikeda, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts., J Biosci Bioeng, doi: 10.1016/j.jbiosc.2014.10.008., 119, 5, 596-603, 2015.05.
45.	Masaki Yamaguchi, Akira Ito, Akihiko Ono, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible gene expression system by applying alternating magnetic field to magnetic nanoparticles, ACS Synthetic Biology, 10.1021/sb4000838, 3, 5, 273-279, 2014.05, [URL], By combining synthetic biology with nanotechnology, we demonstrate remote controlled gene expression using a magnetic field. Magnetite nanoparticles, which generate heat under an alternating magnetic field, have been developed to label cells. Magnetite nanoparticles and heat-induced therapeutic genes were introduced into tumor xenografts. The magnetically triggered gene expression resulted in tumor growth inhibition. This system shows great potential for controlling target gene expression in a space and time selective manner and may be used for remote control of cell functions via gene expression..
46.	Akira Ito, Yasunori Yamamoto, Masanori Sato, Kazushi Ikeda, Masahiro Yamamoto, Hideaki Fujita, Eiji Nagamori, Kawabe Yoshinori, Masamichi Kamihira, Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation, Scientific Reports, 10.1038/srep04781, 4, 2014.04, [URL], Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3â €...V/mm amplitude, 4â €...ms width, and 1â €...Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs..
47.	Masanori Sato, Kazushi Ikeda, Shota Kanno, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Enhancement of contractile force generation of artificial skeletal muscle tissues by mild and transient heat treatment, Current Pharmaceutical Biotechnology, 10.2174/1389201015666140408125231, 14, 13, 1083-1087, 2014.07, [URL], Artificial skeletal muscle tissues composed of cells are expected to be used for applications of regenerative medicine and drug screening. Generally, however, the physical forces generated by tissue-engineered skeletal muscle are lower than those of skeletal muscle tissues found in the body. Local hyperthermia is used for many diseases including muscle injuries. It was recently reported that mild heat treatment improved skeletal muscle functions. In this study, we investigated the effects of mild heat treatment on the tissue-engineered skeletal muscle tissues in vitro. We used magnetite cationic liposomes to label C2C12 myoblast cells magnetically, and constructed densely packed artificial skeletal muscle tissues by using magnetic force. Cell culture at 39°C promoted the differentiation of myoblast cells into myotubes. Moreover, the mild and transient heat treatment improved the contractile properties of artificial skeletal muscle tissue constructs. These findings indicate that the culture method using heat treatment is a useful approach to enhance functions of artificial skeletal muscle tissue..
48.	Ito A, Yamaguchi M, Okamoto N, Sanematsu Y, Kawabe Y, Wakamatsu K, Ito S, Honda H, Kobayashi T, Nakayama E, Tamura Y, Okura M, Yamashita T, Jimbow K, Kamihira M, T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles, Nanomedicine (Lond), doi: 10.2217/nnm.12.142, 8, 6, 891-902, 2013.06.
49.	Kazuhiro Yoshida, Yuya Okuzaki, Ken Ichi Nishijima, Kenji Kyogoku, Takashi Yamashita, Kawabe Yoshinori, Makoto Motono, Masamichi Kamihira, Shinji Iijima, Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan, Cytotechnology, 10.1007/s10616-013-9613-z, 65, 6, 985-992, 2013.12, [URL], The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of α2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serum-derived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood..
50.	Masanori Sato, Akira Ito, Hirokazu Akiyama, Kawabe Yoshinori, Masamichi Kamihira, Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering, Tissue Engineering - Part A., 10.1089/ten.tea.2011.0728, 19, 1-2, 307-315, 2013.01, [URL], 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 B-cell lymphoma 2 (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..
51.	Masaki Yamaguchi, Akira Ito, Noriaki Okamoto, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.05.006, 114, 4, 460-465, 2012.10, [URL], 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..
52.	Kawabe Yoshinori, Hirokatsu Makitsubo, Yujiro Kameyama, Shuohao Huang, Akira Ito, Masamichi Kamihira, Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system, Cytotechnology, 10.1007/s10616-011-9397-y, 64, 3, 267-279, 2012.05, [URL].
53.	Hirokazu Obayashi, Kawabe Yoshinori, Hirokatsu Makitsubo, Ryoko Watanabe, Yujiro Kameyama, Shuohao Huang, Yuta Takenouchi, Akira Ito, Masamichi Kamihira, Accumulative gene integration into a pre-determined site using Cre/loxP, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.10.027, 113, 3, 381-388, 2012.03, [URL].
54.	Yasunori Yamamoto, Akira Ito, Hideaki Jitsunobu, Katsuya Yamaguchi, Kawabe Yoshinori, Hiroshi Mizumoto, Masamichi Kamihira, Hollow fiber bioreactor perfusion culture system for magnetic force-based skeletal muscle tissue engineering, Journal of Chemical Engineering of Japan, 10.1252/jcej.11we237, 45, 5, 348-354, 2012.01, [URL], Large-scale skeletal muscle tissue cultures are often limited by nutrient supplementation and oxygen diffusion. In the present study, we used a hollow-fiber bioreactor system to supply nutrients and oxygen for the cultivation of high cell-density skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique. C2C12 cells, magnetically-labeled with magnetite cationic liposomes (MCLs), were mixed with a type I collagen solution and seeded into the cell culture space of the hollow-fiber bioreactor. A magnet was then placed underneath the bioreactor to accumulate MCL-labeled cells in the space between the hollow fibers by magnetic force. Perfusion culture was performed using a myogenic differentiation medium for 7 d. Histological observation revealed that high cell-dense and viable tissue constructs containing myotubes were successfully formed. Furthermore, muscle-specific proteins, such as myosin heavy chain and tropomyosin, were detected by western blot, indicating that C2C12 cells underwent myogenic differentiation. These findings indicate that the hollow-fiber bioreactor system is an effective approach for the in vitro culture of large skeletal muscle tissue constructs, fabricated by magnetic force-based tissue engineering. &copy 2012 The Society of Chemical Engineer, Japan..
55.	Daisuke Kodama, Daisuke Nishimiya, Ken ichi Nishijima, Yuuki Okino, Yujin Inayoshi, Yasuhiro Kojima, Ken ichiro Ono, Makoto Motono, Katsuhide Miyake, Kawabe Yoshinori, Kenji Kyogoku, Takashi Yamashita, Masamichi Kamihira, Shinji Iijima, Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.10.006, 113, 2, 146-153, 2012.02, [URL], We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to - 3861. bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning - 3861 to - 1569. bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately - 1800. bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (- 2112 to - 1569. bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene..
56.	Hideaki Yamamoto, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors, Biochemical Engineering Journal, 10.1016/j.bej.2011.10.004, 60, 67-73, 2012.01, [URL], Hepatoma cells, which are derived from liver carcinoma, are able to proliferate infinitely under culture conditions. However, the liver functions of hepatoma cells are generally low compared with those of hepatocytes in a liver. Here, we attempted to create genetically engineered hepatoma cells with enhanced liver functions by overexpression of liver-enriched transcription factors (LETFs), which are associated with the transcription of liver-specific genes and hepatic differentiation. For this purpose, genes for eight LETFs, hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β, HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ, were obtained from the mouse liver. Mouse hepatoma Hepa1-6 cells were transduced with retroviral vectors, in which inducible expression cassettes for the LETF genes were introduced. Cell clones with inducible expression of high liver functions were established. Upon overexpression of the LETF genes, cell proliferation ceased and the cells exhibited an epithelial morphology, indicating hepatic maturation of hepatoma cells. This approach for genetic modification of hepatoma cells may be promising for the construction of cells for use in bioartificial liver support systems..
57.	Shuohao Huang, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2011.11.059, 417, 1, 78-83, 2012.01, [URL], Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site..
58.	Akira Ito, Noriaki Okamoto, Masaki Yamaguchi, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator, International Journal of Hyperthermia, 10.3109/02656736.2012.738847, 28, 8, 788-798, 2012.12, [URL], 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..
59.	Kawabe Y, Hayashida Y, Numata K, Harada S, Hayashida Y, Ito A, Kamihira M, Oral immunotherapy for pollen allergy using T-cell epitope-containing egg white derived from genetically manipulated chickens, PLOS ONE, doi:10.1371/journal.pone.0048512, 7, 10, e48512, 2012.10, [URL].
60.	Masanori Sato, Akira Ito, Kawabe Yoshinori, Eiji Nagamori, Masamichi Kamihira, Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.05.007, 112, 3, 273-278, 2011.09, [URL], The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators..
61.	Masanobu Horie, Akira Ito, Takeshi Maki, Kawabe Yoshinori, Masamichi Kamihira, Magnetic separation of cells from developing embryoid bodies using magnetite cationic liposomes, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.04.011, 112, 2, 184-187, 2011.08, [URL], 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 8. d of differentiation using a magnetic separation method with magnetite cationic liposomes..
62.	Yasunori Yamamoto, Akira Ito, Hideaki Fujita, Eiji Nagamori, Kawabe Yoshinori, Masamichi Kamihira, Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique, Tissue Engineering - Part A., 10.1089/ten.tea.2010.0312, 17, 1-2, 107-114, 2011.01, [URL], Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2μN [1.06mN/mm²]). Rheobase and chronaxie of the tissue were determined as 4.45V and 0.72ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies..
63.	Shuohao Huang, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector, Biotechnology and Bioengineering, 10.1002/bit.22863, 107, 4, 717-729, 2010.11, [URL], Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717-729..
64.	Masanobu Horie, Akira Ito, Takehiko Kiyohara, Kawabe Yoshinori, Masamichi Kamihira, E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2010.06.002, 110, 5, 582-587, 2010.11, [URL], Conventionally, embryonic stem (ES) cells are cultured on a cell layer of mouse embryonic fibroblasts (MEFs) as feeder cells to support undifferentiated growth of ES cells. In this study, cell-cell interactions between mouse ES and feeder cells were artificially engineered via an epithelial cell adhesion molecule, E-cadherin, whose expression is considerable in ES cells. Mouse mesenchymal STO and NIH3T3 cells that were genetically engineered to express E-cadherin were used in ES cell cultures as feeder cells. ES cells cultured on the E-cadherin-expressing feeder cells maintained the expression of stem cell markers, alkaline phosphatase (AP), Oct3/4, Nanog and Sox2, and the efficiency of AP-positive colony formation was comparable to MEFs, and much better than parental STO and NIH3T3 cells. Furthermore, ES cells maintained on the E-cadherin-expressing feeder cells possessed the ability to differentiate into the three germ layers both in vitro and in vivo. The results indicated that E-cadherin expression in feeder cells could improve the performance of feeder cells, which may be further applicable to create new artificial feeder cell lines..
65.	Hirokazu Akiyama, Akira Ito, Masanori Sato, Kawabe Yoshinori, Masamichi Kamihira, Construction of cardiac tissue rings using a magnetic tissue fabrication technique, International Journal of Molecular Sciences, 10.3390/ijms11082910, 11, 8, 2910-2920, 2010.08, [URL], Here we applied a magnetic force-based tissue engineering technique to cardiac tissue fabrication. A mixture of extracellular matrix precursor and cardiomyocytes labeled with magnetic nanoparticles was added into a well containing a central polycarbonate cylinder. With the use of a magnet, the cells were attracted to the bottom of the well and allowed to form a cell layer. During cultivation, the cell layer shrank towards the cylinder, leading to the formation of a ring-shaped tissue that possessed a multilayered cell structure and contractile properties. These results indicate that magnetic tissue fabrication is a promising approach for cardiac tissue engineering..
66.	Yujiro Kameyama, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange, Biotechnology and Bioengineering, 10.1002/bit.22619, 105, 6, 1106-1114, 2010.04, [URL].
67.	Carlos Alberto Penno, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens, Transgenic Research, 10.1007/s11248-009-9310-z, 19, 2, 187-195, 2010.04, [URL].
68.	Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles, Journal of Biomedical Materials Research - Part A, 10.1002/jbm.a.32313, 92, 3, 1123-1130, 2010.03, [URL], Development of cell-patterning techniques is a major challenge for the construction of functional tissues and organs in tissue engineering. Recent progress in surface chemistry has enabled spatial control of cell adhesion onto cultural substrates by varying hydrophilicity, for example, by using poly (ethylene glycol) (PEG). In the present study, we developed a novel cell-patterning procedure using PEG-modified magnetite particles (PEG-Mags) and magnetic force. Using an array-patterned magnet, PEG-Mags were magnetically patterned on the surface of a tissue culture dish. The resultant substrate surface consisted of two regions: the PEG-Mag surface that acts as a cell-resistant region and the native substrate surface that promotes cell adhesion. When human keratinocyte HaCaT cells were seeded onto the PEG-Mag-patterned surface, cells adhered only to the native substrate surface, resulting in cell-patterning on the tissue culture dish. The patterned PEG-Mags were then washed away to expose the native substrate surface, and thereafter, when mouse myoblast C2C12 cells were seeded to the dish, cells adhered to the exposed substrate surface, resulting in a patterned coculture of heterotypic cells. Moreover, it is worth noting that the magnetic force-based cell-patterning procedure is not limited by the property of cultural substrate surfaces, and that cell-patterning of mouse fibroblast NIH3T3 cells on a monolayer of HaCaT cells was successfully achieved using PEG-Mags and magnetic force. These results indicate that this procedure provides a novel concept for cell-patterning and may be useful for tissue engineering and cell biology..
69.	Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques, Biomaterials, 10.1016/j.biomaterials.2009.11.017, 31, 6, 1251-1259, 2010.02, [URL], A major limitation in tissue engineering is the insufficient formation of blood vessels in implanted tissues, resulting in reduced cell density and graft size. We report here the fabrication of angiogenic cell sheets using a combination of two magnetic force-based techniques which use magnetite cationic liposomes (MCLs), magnetofection and magnetic cell accumulation. A retroviral vector encoding an expression cassette of vascular endothelial growth factor (VEGF) was labeled with MCLs, to magnetically attract the particles onto a monolayer of mouse myoblast C2C12 cells, for gene delivery. MCL-mediated infection increased transduction efficiency by 6.7-fold compared with the conventional method. During the fabrication of the tissue constructs, MCL-labeled cells were accumulated in the presence of a magnetic field to promote the spontaneous formation of a multilayered cell sheet. VEGF gene-engineered C2C12 (C2C12/VEGF) cell sheets, constructed using both magnetic force-based techniques, were subcutaneously transplanted into nude mice. Histological analyses revealed that on day 14 the C2C12/VEGF cell sheet grafts had produced thick tissues, with a high-cell density, and promoted vascularization. This suggests that the method described here represents a powerful strategy in tissue engineering..
70.	Yasunori Yamamoto, Akira Ito, Masahiro Kato, Kawabe Yoshinori, Kazunori Shimizu, Hideaki Fujita, Eiji Nagamori, Masamichi Kamihira, Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2009.05.019, 108, 6, 538-543, 2009.12, [URL], Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering..
71.	Akira Ito, Hirokazu Akiyama, Yasunori Yamamoto, Kawabe Yoshinori, Masamichi Kamihira, Skeletal muscle tissue engineering using functional magnetite nanoparticles, 20th Anniversary MHS 2009 and Micro-Nano Global COE - 2009 International Symposium on Micro-NanoMechatronics and Human Science 20th Anniversary MHS 2009 and Micro-Nano Global COE - 2009 International Symposium on Micro-NanoMechatronics and Human Science, 10.1109/MHS.2009.5351986, 379-382, 2009.11, [URL], Skeletal muscular tissues were constructed using magnetic force-based tissue engineering (Mag-TE) techniques. Mouse myoblast C2C12 cells labeled with magnetite cationic liposomes (MCLs) were seeded into a well of 24-well ultra-low cell attachment culture plates. When a magnet was positioned underneath the well, cells accumulated evenly onto the culture surface and formed a multilayered cell sheet. Furthermore, because an angiogenic potential of transplants is considered to be important for the long-term maintenance of cell survival and tissue functions, a vascular endothelial growth factor (VEGF) gene-modified C2C12 (C2C12/VEGF) cell sheets were also fabricated by the Mag-TE technique. The secretion level of C2C12/VEGF sheets was 3.0 ng/day, indicating that VEGF gene-expressing cell sheets were successfully fabricated. Since the shape of artificial tissue constructs can be controlled by magnetic force, a cellular string-like assembly was formed by placing a linear-shaped magnetic field concentrator with a magnet. These cellular sheets and strings shrank and did not maintain their shapes for an additional in vitro culture period during myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of well during the fabrication of cell sheets, the cell sheets shrank and formed a ring-like assembly around the plug. After 6-d cultivation of cell rings in differentiation medium, the C2C12 cells differentiated to form multinucleated myotubes. Thus, these procedures can provide a novel strategy for skeletal muscular tissue engineering..
72.	Akira Ito, Tetsuya Takahashi, Kawabe Yoshinori, Masamichi Kamihira, Human beta defensin-3 engineered keratinocyte sheets constructed by a magnetic force-based tissue engineering technique, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2009.04.004, 108, 3, 244-247, 2009.09, [URL], A multilayered keratinocyte sheet overexpressing human beta defensin-3 (HBD-3) gene was prepared by the magnetic force-based tissue engineering technique. The cell sheet demonstrated significant antimicrobial activity, indicating that the therapeutic introduction of HBD-3 gene into cell sheets may provide a new gene therapy strategy for infectious diseases..
73.	Akira Ito, Hideaki Jitsunobu, Kawabe Yoshinori, Hiroyuki Ijima, Masamichi Kamihira, Magnetic separation of cells in coculture systems using magnetite cationic liposomes., Tissue Engineering - Part C: Methods, 10.1089/ten.tec.2008.0496, 15, 3, 413-423, 2009.09, [URL], In tissue engineering, coculture systems have been employed for two major purposes: (1) construction of tissue and organ substitutes (e.g., coculture of parenchymal and nonparenchymal cells in liver tissue engineering) and (2) maintenance of cellular functions (e.g., coculture of embryonic stem cells with embryonic fibroblasts as the feeder cells). For the characterization and recovery of specific cell types, however, target cells have to be isolated from other cells. We report here a novel magnetic separation method to isolate target cells in coculture systems. In this method, target cells were cocultured with nontarget cells labeled with magnetite cationic liposomes (MCLs). Thus, when necessary, the MCL-labeled nontarget cells were magnetically removed from the coculture, resulting in negative isolation of the target cells. As the separation models, three deferent types of coculture systems were examined: rat hepatocytes with various MCL-labeled cells (mouse NIH3T3, STO, or human umbilical vein endothelial cells), human keratinocyte HaCaT cells with MCL-labeled NIH3T3 cells, and mouse embryonic stem cells with MCL-labeled STO cells. In these cocultures, target cells were separated with 94% purity and 98%recovery yield on average. This technique provides a promising approach to isolate and recover target cells for further analysis and application..
74.	Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique, Biomedical Microdevices, 10.1007/s10544-009-9284-x, 11, 4, 713-721, 2009.08, [URL], We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering..
75.	Masamichi Kamihira, Kawabe Yoshinori, Takuya Shindo, Ken ichiro Ono, Kazuhisa Esaka, Takashi Yamashita, Ken ichi Nishijima, Shinji Iijima, Production of chimeric monoclonal antibodies by genetically manipulated chickens, Journal of Biotechnology, 10.1016/j.jbiotec.2009.02.022, 141, 1-2, 18-25, 2009.04, [URL], Genetically manipulated chickens producing chimeric monoclonal antibodies were generated by injecting retroviral vectors encoding genes for the heavy and light chains of antibodies into developing embryos. The transgene was detected in all chickens that hatched, and they stably produced the chimeric antibodies in their serum. After sexual maturation, the antibodies were also produced in eggs laid by the manipulated hens. The stable antibody production was observed both in egg white and yolk throughout the breeding period. The chimeric antibodies produced by the chickens were properly assembled and exhibited antigen-binding activities. Furthermore, we characterized the structures of the N-linked oligosaccharide chains added to the Fc-region of the recombinant antibodies produced in the serum, egg white and yolk of the chickens..
76.	Akira Ito, Tetsuya Takahashi, Yujiro Kameyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic concentration of a retroviral vector using magnetite cationic liposomes, Tissue Engineering - Part C: Methods, 10.1089/ten.tec.2008.0275, 15, 1, 57-64, 2009.03, [URL], For tissue engineering purposes, retroviral vectors represent an efficient method of delivering exogenous genes such as growth factors to injured tissues because gene-transduced cells can produce stable and constant levels of the gene product. However, retroviral vector technology suffers from low yields. In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with cationic liposomes were added to a solution containing a retroviral vector pseudotyped with vesicular stomatitis virus glycoprotein. The magnetic particles that captured the viral vectors were collected using a magnetic force and seeded into mouse neuroblastoma Neuro2a cells. The viral titer was up to 55 times greater (up to 3 × 10⁸ infectious units/mL). Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this technique provides a promising approach to capturing and concentrating viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specific injured site by applying a magnetic field..
77.	Kawabe Yoshinori, Tsutomu Naka, Hiroyuki Komatsu, Ken Ichi Nishijima, Shinji Iijima, Masamichi Kamihira, Retroviral Gene Transduction into Chicken Embryo Gonads through Blood Circulation, Journal of Bioscience and Bioengineering, 10.1263/jbb.106.598, 106, 6, 598-601, 2008.12, [URL].
78.	Akira Ito, Tetsuya Takahashi, Yujiro Kameyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic manipulation of a retroviral vector using magnetite cationic liposomes, 2008 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2008, with Symposium on "COE for Education and Research of Micro-Nano Mechatronics", Symposium on "System Cell Engineering by Multi-scale Manipulation" 2008 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2008, with Symposium on "COE for Education and Research of Micro-Nano Mechatronics", 10.1109/MHS.2008.4752479, 367-371, 2008.11, [URL], In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors in order to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with Cationic liposomes were added to a solution containing a retroviral vector. The magnetic particles which captured the viral vectors were collected by a magnetic force, and seeded into target cells. The viral titer increased up to 55-fold and 3 x 10⁸ IU/mL. Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this new technique provides a promising approach to capture and concentrate viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specifically injured site by applying a magnetic field..
79.	Yujiro Kameyama, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein, Journal of Virological Methods, 10.1016/j.jviromet.2008.06.013, 153, 1, 49-54, 2008.10, [URL], Gammaretroviral and lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) have been used for stable gene transfer because of their broad host range and high mechanical strength. In the present study, an expression plasmid for chimeric VSV-G, consisting of a ZZ fragment derived from Staphylococcal protein A fused to the N-terminus of VSV-G (ZZ-VSV-G), was constructed to produce viral vectors capable of antibody-dependent gene transduction. Gammaretroviral (based on mouse stem cell virus, MSCV) and lentiviral (based on human immunodeficiency virus type 1, HIV-1) vectors pseudotyped with ZZ-VSV-G were produced without the loss of antibody-binding activity. The production of infectious viral particles was promoted by the addition of an expression plasmid for native VSV-G and antibody-dependent gene transduction was achieved using plates coated with antibodies. This system may be useful for the genetic transduction of cells expressing specific proteins on their surface, and for screening of antibodies specific for cell surface receptors..
80.	Akira Ito, Takehiko Kiyohara, Kawabe Yoshinori, Hiroyuki Ijima, Masamichi Kamihira, Enhancement of cell function through heterotypic cell-cell interactions using E-cadherin-expressing NIH3T3 cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.105.679, 105, 6, 679-682, 2008.06, [URL], 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..
81.	Kenji Kyogoku, Kazuhiro Yoshida, Hiroyuki Watanabe, Takashi Yamashita, Kawabe Yoshinori, Makoto Motono, Ken Ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens, Journal of Bioscience and Bioengineering, 10.1263/jbb.105.454, 105, 5, 454-459, 2008.05, [URL], We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-α toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens..
82.	Daisuke Kodama, Daisuke Nishimiya, Ken ichi Iwata, Kazuhisa Yamaguchi, Kazuhiro Yoshida, Kawabe Yoshinori, Makoto Motono, Hiroyuki Watanabe, Takashi Yamashita, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of human erythropoietin by chimeric chickens, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2008.01.020, 367, 4, 834-839, 2008.03, [URL], The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells..
83.	Akira Ito, Hideaki Jitsunobu, Kawabe Yoshinori, Masamichi Kamihira, Construction of Heterotypic Cell Sheets by Magnetic Force-Based 3-D Coculture of HepG2 and NIH3T3 Cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.371, 104, 5, 371-378, 2007.11, [URL], Heterotypic 3-D coculture is essential to mimic tissues and organs, because cell-cell interaction between various types of cells is believed to be important for the activation of cellular functions. In this study, magnetic force was applied to construct a 3-D coculture system of HepG2 and NIH3T3 cells as a model of hepatocytes and mesenchymal cells. Magnetite cationic liposomes (MCLs) were used to label target cells. NIH3T3 cells labeled with MCLs were seeded onto ultralow-attachment plates, whose surface is composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. When a magnet was placed under the plate, cells accumulated on the bottom of the well. After a 24-h incubation period, the cells formed a multilayered cell sheet, which contained the major mesenchymal extracellular matrix (ECM) components (fibronectin and type I collagen), suggesting that the use of stromal NIH3T3 cells gave sufficient strength to cell sheets. Both NIH3T3 and HepG2 cells were labeled with MCLs, and cocultured by two methods: NIH3T3 cell sheets were constructed and HepG2 cells were subsequently seeded onto NIH3T3 cell sheets, and then allowed to form layered cell sheets by applying magnetic force; or NIH3T3 and HepG2 cells were mixed and then allowed to form mixed cell sheets by applying magnetic force. These heterotypic multilayered cell sheets were successfully constructed and an enhanced albumin secretion by HepG2 cells was observed. These results suggest that the new tissue engineering technique using magnetite nanoparticles and magnetic force, to which we refer to as magnetic force-based tissue engineering (Mag-TE), is a promising approach to construct multilayered cell sheets consisting of heterotypic cocultured cells..
84.	Akira Ito, Hirokazu Akiyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic force-based cell patterning using Arg-Gly-Asp (RGD) peptide-conjugated magnetite cationic liposomes, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.288, 104, 4, 288-293, 2007.10, [URL], Micropatterning of target cells is highly desired for tissue engineering and cell biology. Although recent progress in surface chemistry has enabled the spatial control of cell adhesion onto substrates, conventional methods usually require specialized devices and time-consuming processes to fabricate the substrate. In this study, we demonstrate a simple and rapid cell-patterning procedure using magnetite nanoparticles and magnetic force. To label the target cells magnetically, magnetite nanoparticles were encapsulated in cationic liposomes (magnetite cationic liposomes; MCLs). To promote cell attachment, an Arg-Gly-Asp (RGD)-motif-containing peptide was coupled to the phospholipid of MCLs (RGD-MCLs). A human keratinocyte cell line, HaCaT, which has a high anchorage dependency, was used as a model. The RGD-MCLs were added to an ultralow-attachment plate, whose culture surface is modified with a covalently bound hydrogel layer that is hydrophilic and neutrally charged, and then HaCaT cells were seeded to the plates. The RGD-MCLs induced cell adhesion, spreading, cytoskeletal organization, and fibronectin expression. When steel plates with a 200 μm width placed on a magnet were set under a culture surface, magnetically labeled cells aligned on the surface where the steel plate was positioned, resulting in cell patterning. Furthermore, various cell patterns using a computer-aided design were successfully fabricated. These results suggest that cell patterning using RGD-MCLs is a promising approach to tissue engineering and studies in cell biology..
85.	Kawabe Yoshinori, Tsutomu Naka, Naoko Ando-Noumi, Hiroyuki Matsumoto, Ken ichiro Ono, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.518, 102, 6, 518-523, 2006.12, [URL].
86.	Kawabe Yoshinori, Masamichi Kamihira, Ken ichiro Ono, Kenji Kyogoku, Ken ichi Nishijima, Shinji Iijima, Production of scFv-Fc fusion protein using genetically manipulated quails, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.297, 102, 4, 297-303, 2006.10, [URL].
87.	Akitsu Hotta, Yoshikazu Saito, Kenji Kyogoku, Kawabe Yoshinori, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Characterization of transient expression system for retroviral vector production, Journal of Bioscience and Bioengineering, 10.1263/jbb.101.361, 101, 4, 361-368, 2006.04, [URL], The production of retroviral vectors using a transient expression system has been improved to obtain a high-titer virus preparation that is difficult to produce using packaging cell lines due to the cytotoxic or cytostatic effect of transgenes. Here, we used one such production method, the so-called Q-vector system, and examined its potential for virus production. The Q-vector system could produce a similar level of viral vectors compared with the packaging cell system but the production seemed to depend on the size and nature of transgenes. In the process of investigation of the quantitative difference in viral components between the transient expression system and the packaging cell system, we found that the Q-vector system could express higher amounts of viral RNA and proteins compared with the packaging cell system. However, this did not lead to a higher virus titer compared with that produced by the packaging cell system. This suggests that retroviral RNA transcribed from the plasmid in the transient system seemed to be used mainly for translation and only some of the RNA molecules were packaged in viral particles..
88.	Akitsu Hotta, Masamichi Kamihira, Kanako Itoh, Mahboob Morshed, Kawabe Yoshinori, Ken Ichiro Ono, Hiroyuki Matsumoto, Ken Ichi Nishijima, Shinji Iijima, Production of anti-CD2 chimeric antibody by recombinant animal cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.98.298, 98, 4, 298-303, 2004.01, [URL], Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H- and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5′Cap-and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5′Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein..
89.	Yoshinori Kawabe, Akitsu Hotta, Ken-ichiro Ono, Kazuhisa Esaka, Ken-ichi Nishijima, Masamichi Kamihira, and Shinji Iijima, Production of chimeric antibodies by transgenic chicken bioreactor, Animal Cell Technology: Basic & Applied Aspects, https://doi.org/10.1007/1-4020-4457-7_42, 14, 307-314, 2006.05, [URL].

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