| 1. |
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, It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.. |
| 2. |
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, DOI:10.1002/biot.201800063, 13, 7, e1800063, 2018.05. |
| 3. |
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, J Biosci Bioeng, doi: 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05. |
| 4. |
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. |
| 5. |
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, We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production.. |
| 6. |
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, Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.. |
| 7. |
Yoshinori Kawabe, 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., J Biosci Bioeng., 123(3):390-397, doi: 10.1016/j.jbiosc.2016.10.011., 2016.11. |
| 8. |
Yoshinori Kawabe, 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., Biotechnol Bioeng., 10.1002/bit.25923., 113, 7, 1600-1610, 2016.01. |
| 9. |
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. |
| 10. |
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, e48512, 2012.10. |
| 11. |
Obayashi H, Kawabe Y, Makitsubo H, Watanabe R, Kameyama Y, Huang S, Takenouchi Y, Ito A, Kamihira M, Accumulative gene integration into a pre-determined site using Cre/loxP., J Biosci Bioeng, doi:10.1016/j.jbiosc.2011.10.027, 113, 3, 381-388, 2012.05. |
| 12. |
Kawabe Yoshinori, Makitsubo H, Kameyama Y, Huang S, 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, DOI: 10.1007/s10616-011-9397-y, 64(3)、267-279, 2012.05. |
| 13. |
Yamamoto H, Kawabe Y, Ito A, Kamihira M, Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors., Biochem Eng J, doi:10.1016/j.bej.2011.10.004, 60, 15, 67-73, 2012.01. |
| 14. |
Huang S, Kawabe Y, Ito A, Kamihira M, Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19., Biochem Biophys Res Commun, doi:10.1016/j.bbrc.2011.11.059, 417, 1, 78-83, 2012.01. |
| 15. |
Huang S, Kawabe Y, Ito A, Kamihira M , Cre recombinase-mediated site-specific modification of a celluar genome using an integrase-defective retroviral vector, Biotechnol Bioeng, 107: 717-29, 2010.11. |
| 16. |
Penno CA, Kawabe Y, Ito A, Kamihira M., Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens, Transgenic Res. , 19(2):187-95., 2010.04. |
| 17. |
Kameyama Y, Kawabe Y, Ito A, Kamihira M., An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange., Biotechnol Bioeng. , 105(6):1106-14., 2010.04. |
| 18. |
Kamihira M, Kawabe Y, Shindo T, Ono K, Esaka K, Yamashita T, Nishijima K, Iijima S, Production of chimeric monoclonal antibodies by genetically manipulated chickens, J Biotechnol. , 141(1-2):18-25., 2009.04. |
| 19. |
Kawabe Y, Naka T, Komatsu H, Nishijima K, Iijima S, Kamihira M., Retroviral gene transduction into chicken embryo gonads through blood circulation, J Biosci Bioeng, 106(6):598-601, 2008.12. |
| 20. |
Kameyama Y, Kawabe Y, Ito A, Kamihira M, Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein, J Virol Methods, doi:10.1016/j.jviromet.2008.06.013, 153, 1, 49-54, 2008.06. |
| 21. |
Kawabe Y, Naka T, Ando-Noumi N, Matsumoto H, Ono K, Nishijima K, Kamihira M, Iijima S, Transport of human Immunoglobulin G and Fc-Fusion Proteins to Chicken Egg Yolk, J Biosci Bioen, doi:10.1263/jbb.102.518, 102, 6, 518-523, 2006.12. |
| 22. |
Kawabe Y, Kamihira M, Ono K, Kyogoku K, Nishijima K, Iijima S, Production of scFv-Fc Fusion Protein by Genetically Manipulated Quails, J Biosci Bioeng , doi:10.1263/jbb.102.297, 102, 4, 297-303, 2006.10. |
| 23. |
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, 14,307-314, 2006.05. |
