| 1. |
Fluctuation and Biological Function. |
| 2. |
Comprehensive Analysis of Transcriptional Bursting in Mammalian Cells. |
| 3. |
Functional analysis using CRISPR library. |
| 4. |
Hiroshi Ochiai, Takashi Yamamoto, Genome editing using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System, 10.1007/978-4-431-55227-7_1, pp.3-24, 2015.01, Targetable nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas), induce DNA double- strand breaks (DSBs) into user-defined sites. DSBs are immediately repaired through the evolutionarily conserved pathways of error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR). With the utilization of these repair processes, researchers have been able to disrupt specific genes, add exogenous DNA elements into intended genomic sites, introduce single-nucleotide substitutions, and perform many other applications. Consequently, this “genome editing” technology has revolutionized the life science field. In addition, this technology has the potential to improve agricultural products and be applicable to therapeutic use. Here, we will introduce a brief history of targetable nuclease-mediated genome editing and the applications of the tools that the technology provides. In this chapter, we will primarily focus on ZFNs and TALENs, which are artificial proteins composed of a specific DNA-binding domain and a restriction enzyme FokI DNAcleavage domain. We will also review the properties and construction methods of these nucleases.. |
| 5. |
Identification of mutation causing cancer-prone hereditary disease using a novel method for the introduction of single-nucleotide substitution. |
| 6. |
Tetsushi Sakuma, Sayaka Hosoi, Knut Woltjen, Ken-ichi Suzuki, Keiko Kashiwagi, Housei Wada, Hiroshi Ochiai, Tatsuo Miyamoto, Narudo Kawai, Yasunori Sasakura, Shinya Matsuura, Yasushi Okada, Atsuo Kawahara, Shigeo Hayashi, Takashi Yamamoto, Efficient TALEN construction and evaluation methods for human cell and animal applications, GENES TO CELLS, 10.1111/gtc.12037, Vol.18, No.4, pp.315-326, 2013.04, Transcription activatorlike effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.. |
| 7. |
Quantitative Imaging of Gene Expression Using Genome Editing Technology. |
| 8. |
落合 博, 佐久間哲史, 松浦伸也, 山本 卓, TALE nuclease (TALEN)を用いた培養細胞におけるゲノム編集, 実験医学, Vol.31, No.1, pp.95-100, 2013.01. |
