Efficient <scp>TALEN</scp> construction and evaluation methods for human cell and animal applications
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- Tetsushi Sakuma
- Department of Mathematical and Life Sciences Graduate School of Science Hiroshima University 1‐3‐1 Kagamiyama Higashi‐Hiroshima Hiroshima 739‐8526 Japan
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- Sayaka Hosoi
- Department of Mathematical and Life Sciences Graduate School of Science Hiroshima University 1‐3‐1 Kagamiyama Higashi‐Hiroshima Hiroshima 739‐8526 Japan
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- Knut Woltjen
- Center for iPS Cell Research and Application (CiRA) Kyoto University 53 Shogoin‐Kawahara‐cho Sakyo‐ku Kyoto 606‐8507 Japan
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- Ken‐ichi Suzuki
- Department of Mathematical and Life Sciences Graduate School of Science Hiroshima University 1‐3‐1 Kagamiyama Higashi‐Hiroshima Hiroshima 739‐8526 Japan
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- Keiko Kashiwagi
- Institute for Amphibian Biology Graduate School of Science Hiroshima University 1‐3‐1 Kagamiyama Higashi‐Hiroshima Hiroshima 739‐8526 Japan
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- Housei Wada
- Laboratory for Morphogenetic Signaling RIKEN Center for Developmental Biology 2‐2‐3 Minatojima‐minamimachi Chuo‐ku Kobe Hyogo 650‐0047 Japan
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- Hiroshi Ochiai
- Department of Genetics and Cell Biology Research Institute for Radiation Biology and Medicine Hiroshima University 1‐2‐3 Kasumi Minami‐ku Hiroshima 734‐8553 Japan
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- Tatsuo Miyamoto
- Department of Genetics and Cell Biology Research Institute for Radiation Biology and Medicine Hiroshima University 1‐2‐3 Kasumi Minami‐ku Hiroshima 734‐8553 Japan
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- Narudo Kawai
- Department of Biology Research and Education Center for Natural Sciences Keio University 4‐1‐1 Hiyoshi Kohoku‐ku Yokohama 223‐8521 Japan
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- Yasunori Sasakura
- Shimoda Marine Research Center University of Tsukuba 5‐10‐1 Shimoda Shizuoka 415‐0025 Japan
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- Shinya Matsuura
- Department of Genetics and Cell Biology Research Institute for Radiation Biology and Medicine Hiroshima University 1‐2‐3 Kasumi Minami‐ku Hiroshima 734‐8553 Japan
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- Yasushi Okada
- Quantitative Biology Center RIKEN 6‐2‐3 Furuedai Suita Osaka 565‐0874 Japan
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- Atsuo Kawahara
- Quantitative Biology Center RIKEN 6‐2‐3 Furuedai Suita Osaka 565‐0874 Japan
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- Shigeo Hayashi
- Laboratory for Morphogenetic Signaling RIKEN Center for Developmental Biology 2‐2‐3 Minatojima‐minamimachi Chuo‐ku Kobe Hyogo 650‐0047 Japan
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- Takashi Yamamoto
- Department of Mathematical and Life Sciences Graduate School of Science Hiroshima University 1‐3‐1 Kagamiyama Higashi‐Hiroshima Hiroshima 739‐8526 Japan
説明
<jats:p>Transcription activator–like 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/<jats:italic>in vitro</jats:italic> 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 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or <jats:styled-content style="fixed-case">mRNA</jats:styled-content> synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, <jats:italic>Drosophila melanogaster</jats:italic>,<jats:italic> Danio rerio</jats:italic> and <jats:italic>Xenopus laevis,</jats:italic> 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.</jats:p>
収録刊行物
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- Genes to Cells
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Genes to Cells 18 (4), 315-326, 2013-02-06
Wiley
