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A novel MITF-DNA binding inhibitor for depigmentation in melanocytes
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Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20
References 1. Sethe, S., Scutt, A., Stolzing, A.: Aging of mesenchymal stem cells, Ageing Res. Rev., 5, 91-116 (2006). 2. Simm, A., Nass, N., Bartling, B., Hofmann, B., Silber, R. E., Navarrete Santos, A.: Potential biomarkers of ageing, Biol. Chem., 389, 257-265 (2008). 3. Gerke, V., Creutz, C. E., Moss, S. E.: Annexins: linking Ca2+ signalling to membrane dynamics, Nat. Rev. Mol. Cell. Biol., 6, 449-61 (2005).
doi:10.1016/j.jbiosc.2009.08.059
AN-P30 Accumulative gene integration technique using recombinase mediate cassette exchange Yujiro Kameyama, Yoshinori Kawabe, Akira Ito, and Masamichi Kamihira Kyushu University, Fukuoka, Japan The gene amplification technique on cellular genome is an essential process for the establishment of producer cells in biopharmaceutical production using recombinant mammalian cells because higher productivity is required to reduce the production cost. For the amplification of target gene introduced to chromosome, the concentration of drugs according to the selection marker gene is step-wisely increased during the culture. However, the selection process of producer clones is time-consuming and laborious with uncertainty. On the other hand, Cre-loxP system has been used as a powerful tool for site-specific recombination both in vitro and in vivo. In recent years, it was reported that the recombination reaction can be controlled by using mutated loxPs: the irreversible integration reaction is promoted using armmutated loxPs [1], whereas the recombination specificity is restricted using spacer-mutated loxPs [2]. Recently, we developed an accumulative gene integration system using Cre recombinase and mutated loxPs combining arm and spacer mutations, in which Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into the loxP target sites. A series of integration reactions of target genes occurred between plasmids and on the genome of CHO cells. The gene integration system provides a novel strategy for gene amplification on the genome and for biological analyses of gene functions by genetic modification of cells and organisms. References 1. Oberdoerffer, P., Otipoby, K. L., Maruyama, M., and Rajewsky, K.: Undirecional Cremediated genetic inversion in mice using the mutant loxP pair lox66/71, Nucleic Acids Res., 31, e140 (2003). 2. Missirlis, P. I., Smalius, D. E., and Holt, R. A.: A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination, BMC Genomics, 7, 73 (2006).
doi:10.1016/j.jbiosc.2009.08.488
AN-P31 A novel MITF-DNA binding inhibitor for depigmentation in melanocytes Jimin Um, SunA Yoon, Jihyuk Yu, Yuri Lee, Jin Ling, Dung Hoang Nguyen, Hyang-Bok Lee, and Eun-Ki Kim Inha University, Incheon, Republic of Korea
Skin color is determined by the amount of melanin and tyrosinase is a key enzyme of melanin synthesis. MITF (microphthalmiaassociated transcription factor) belongs to the basic helix-loop-helixzip family of transcription factors and is the major regulator of tyrosinase and other related enzymes(TRPs). To screen depigmenting agents by HTS(high-throughput screening), a protein chip containing recombinant MITF protein was constructed. Based on the computersimulated structure, 27 chemicals were selected and were investigated as MITF-DNA binding inhibitors. Among them, compound #18 was found to show the most potent inhibitory activity against MITF-DNA binding. To confirm its inhibitory activity against MITF-DNA binding, an Electrophoretic Mobility Shift Assay (EMSA) was performed. As a result, intensities of MITF-DNA binding bands were dose dependently reduced by compound #18. In addition, to investigate specific inhibition of MITF-DNA binding by compound #18, nuclear extracts of fibroblasts and melanocytes were used for EMSA. Compound #18 did not show any effect on fibroblasts. Therefore, compound #18 could be used as a specific MITF-DNA binding inhibitor on melanocytes. References 1. Saito, H., Yasumoto, K., Takeda, K., Takahashi, K., Yamamoto, H., Shibahara, S.: Microphthalmia-associated transcription factor in the Wnt signaling pathway. Pigment Cell Res., 16(3), 261-5 (2003). 2. Amae, S., Fuse, N., Yasumoto, K., Sato, S., Yajima, I., Yamamoto, H., Udono, T., Durlu, Y. K., Tamai, M., Takahashi, K., Shibahara, S.: Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium. Biochem. Biophys. Res. Commun., 247, 710-715 (1998).
doi:10.1016/j.jbiosc.2009.08.489
AN-P33 Development of mammalian factors-free medium for cell culture by using silk protein sericin Masahiro Kobayashi,1 Akiko Sakuma,2 Yoshihiro Kunitomi,3 Masahiro Sasaki,3 Hideyuki Yamada,3 and Satoshi Terada1 University of Fukui ,Department of Applied Chemistry and Biotechnology, Fukui, Japan 1 JST Satellite Shiga, Otsu, Japan 2 and SEIREN CO. LTD., Fukui, Japan 3 Fetal bovine serum and factors of mammalian origin are extensively used in cell culture for regenerative medicine and protein pharmaceutical production. But those factors are not preferred because of the risk of infectious disease and so on. In this study, we aimed to develop serum-free medium that do not contain any factors of mammalian origin. For the purpose, we used sericin hydrolycate, which is derived from silkworm and has mitogenic activity, as alternative to the factors derived from mammal so as to construct culture medium. As basal medium, Daigo's T Medium (Nihon Seiyaku) was selected since it is rich in nutrients; and CHO-DP12 cell line was used to assay since CHO cells are extensively used as host cells to produce protein pharmaceuticals. In order to develop a mammalian factors-free medium, recombinant insulin, recombinant transferrin, recombinant lactoferrin, ethanolamine or sodium selenite, as well as sericin, were supplemented into the basal medium and the CHO cell line was cultured. Among them, sericin, insulin, ethanolamine and sodium selenite successfully improved the cell culture, while transferrin or lactoferrin failed to improve. With those effective supplements, novel mammalian factors-free medium was constructed and named SericinGIT medium. The novel medium was compared with several serumfree media on the market. It was superior to the commercial serum-
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20
References 1. Sethe, S., Scutt, A., Stolzing, A.: Aging of mesenchymal stem cells, Ageing Res. Rev., 5, 91-116 (2006). 2. Simm, A., Nass, N., Bartling, B., Hofmann, B., Silber, R. E., Navarrete Santos, A.: Potential biomarkers of ageing, Biol. Chem., 389, 257-265 (2008). 3. Gerke, V., Creutz, C. E., Moss, S. E.: Annexins: linking Ca2+ signalling to membrane dynamics, Nat. Rev. Mol. Cell. Biol., 6, 449-61 (2005).
doi:10.1016/j.jbiosc.2009.08.059
AN-P30 Accumulative gene integration technique using recombinase mediate cassette exchange Yujiro Kameyama, Yoshinori Kawabe, Akira Ito, and Masamichi Kamihira Kyushu University, Fukuoka, Japan The gene amplification technique on cellular genome is an essential process for the establishment of producer cells in biopharmaceutical production using recombinant mammalian cells because higher productivity is required to reduce the production cost. For the amplification of target gene introduced to chromosome, the concentration of drugs according to the selection marker gene is step-wisely increased during the culture. However, the selection process of producer clones is time-consuming and laborious with uncertainty. On the other hand, Cre-loxP system has been used as a powerful tool for site-specific recombination both in vitro and in vivo. In recent years, it was reported that the recombination reaction can be controlled by using mutated loxPs: the irreversible integration reaction is promoted using armmutated loxPs [1], whereas the recombination specificity is restricted using spacer-mutated loxPs [2]. Recently, we developed an accumulative gene integration system using Cre recombinase and mutated loxPs combining arm and spacer mutations, in which Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into the loxP target sites. A series of integration reactions of target genes occurred between plasmids and on the genome of CHO cells. The gene integration system provides a novel strategy for gene amplification on the genome and for biological analyses of gene functions by genetic modification of cells and organisms. References 1. Oberdoerffer, P., Otipoby, K. L., Maruyama, M., and Rajewsky, K.: Undirecional Cremediated genetic inversion in mice using the mutant loxP pair lox66/71, Nucleic Acids Res., 31, e140 (2003). 2. Missirlis, P. I., Smalius, D. E., and Holt, R. A.: A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination, BMC Genomics, 7, 73 (2006).
doi:10.1016/j.jbiosc.2009.08.488
AN-P31 A novel MITF-DNA binding inhibitor for depigmentation in melanocytes Jimin Um, SunA Yoon, Jihyuk Yu, Yuri Lee, Jin Ling, Dung Hoang Nguyen, Hyang-Bok Lee, and Eun-Ki Kim Inha University, Incheon, Republic of Korea
Skin color is determined by the amount of melanin and tyrosinase is a key enzyme of melanin synthesis. MITF (microphthalmiaassociated transcription factor) belongs to the basic helix-loop-helixzip family of transcription factors and is the major regulator of tyrosinase and other related enzymes(TRPs). To screen depigmenting agents by HTS(high-throughput screening), a protein chip containing recombinant MITF protein was constructed. Based on the computersimulated structure, 27 chemicals were selected and were investigated as MITF-DNA binding inhibitors. Among them, compound #18 was found to show the most potent inhibitory activity against MITF-DNA binding. To confirm its inhibitory activity against MITF-DNA binding, an Electrophoretic Mobility Shift Assay (EMSA) was performed. As a result, intensities of MITF-DNA binding bands were dose dependently reduced by compound #18. In addition, to investigate specific inhibition of MITF-DNA binding by compound #18, nuclear extracts of fibroblasts and melanocytes were used for EMSA. Compound #18 did not show any effect on fibroblasts. Therefore, compound #18 could be used as a specific MITF-DNA binding inhibitor on melanocytes. References 1. Saito, H., Yasumoto, K., Takeda, K., Takahashi, K., Yamamoto, H., Shibahara, S.: Microphthalmia-associated transcription factor in the Wnt signaling pathway. Pigment Cell Res., 16(3), 261-5 (2003). 2. Amae, S., Fuse, N., Yasumoto, K., Sato, S., Yajima, I., Yamamoto, H., Udono, T., Durlu, Y. K., Tamai, M., Takahashi, K., Shibahara, S.: Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium. Biochem. Biophys. Res. Commun., 247, 710-715 (1998).
doi:10.1016/j.jbiosc.2009.08.489
AN-P33 Development of mammalian factors-free medium for cell culture by using silk protein sericin Masahiro Kobayashi,1 Akiko Sakuma,2 Yoshihiro Kunitomi,3 Masahiro Sasaki,3 Hideyuki Yamada,3 and Satoshi Terada1 University of Fukui ,Department of Applied Chemistry and Biotechnology, Fukui, Japan 1 JST Satellite Shiga, Otsu, Japan 2 and SEIREN CO. LTD., Fukui, Japan 3 Fetal bovine serum and factors of mammalian origin are extensively used in cell culture for regenerative medicine and protein pharmaceutical production. But those factors are not preferred because of the risk of infectious disease and so on. In this study, we aimed to develop serum-free medium that do not contain any factors of mammalian origin. For the purpose, we used sericin hydrolycate, which is derived from silkworm and has mitogenic activity, as alternative to the factors derived from mammal so as to construct culture medium. As basal medium, Daigo's T Medium (Nihon Seiyaku) was selected since it is rich in nutrients; and CHO-DP12 cell line was used to assay since CHO cells are extensively used as host cells to produce protein pharmaceuticals. In order to develop a mammalian factors-free medium, recombinant insulin, recombinant transferrin, recombinant lactoferrin, ethanolamine or sodium selenite, as well as sericin, were supplemented into the basal medium and the CHO cell line was cultured. Among them, sericin, insulin, ethanolamine and sodium selenite successfully improved the cell culture, while transferrin or lactoferrin failed to improve. With those effective supplements, novel mammalian factors-free medium was constructed and named SericinGIT medium. The novel medium was compared with several serumfree media on the market. It was superior to the commercial serum-