Efficient TALEN construction and evaluation methods for human cell and animal applications

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

Research output: Contribution to journalArticle

143 Citations (Scopus)

Abstract

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/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 1 week. 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.

Original languageEnglish
Pages (from-to)315-326
Number of pages12
JournalGenes to Cells
Volume18
Issue number4
DOIs
Publication statusPublished - 2013 Apr

Fingerprint

Induced Pluripotent Stem Cells
Xenopus laevis
Zebrafish
Transcription Activator-Like Effector Nucleases
Drosophila melanogaster
Transfection
Cultured Cells
Embryonic Structures
Yeasts
Genome
Messenger RNA
Proteins
In Vitro Techniques
Transcription Activator-Like Effectors

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Sakuma, T., Hosoi, S., Woltjen, K., Suzuki, K. I., Kashiwagi, K., Wada, H., ... Yamamoto, T. (2013). Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes to Cells, 18(4), 315-326. https://doi.org/10.1111/gtc.12037

Efficient TALEN construction and evaluation methods for human cell and animal applications. / Sakuma, Tetsushi; Hosoi, Sayaka; Woltjen, Knut; Suzuki, Ken Ichi; Kashiwagi, Keiko; Wada, Housei; Ochiai, Hiroshi; Miyamoto, Tatsuo; Kawai, Narudo; Sasakura, Yasunori; Matsuura, Shinya; Okada, Yasushi; Kawahara, Atsuo; Hayashi, Shigeo; Yamamoto, Takashi.

In: Genes to Cells, Vol. 18, No. 4, 04.2013, p. 315-326.

Research output: Contribution to journalArticle

Sakuma, T, Hosoi, S, Woltjen, K, Suzuki, KI, Kashiwagi, K, Wada, H, Ochiai, H, Miyamoto, T, Kawai, N, Sasakura, Y, Matsuura, S, Okada, Y, Kawahara, A, Hayashi, S & Yamamoto, T 2013, 'Efficient TALEN construction and evaluation methods for human cell and animal applications', Genes to Cells, vol. 18, no. 4, pp. 315-326. https://doi.org/10.1111/gtc.12037
Sakuma T, Hosoi S, Woltjen K, Suzuki KI, Kashiwagi K, Wada H et al. Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes to Cells. 2013 Apr;18(4):315-326. https://doi.org/10.1111/gtc.12037
Sakuma, Tetsushi ; Hosoi, Sayaka ; Woltjen, Knut ; Suzuki, Ken Ichi ; Kashiwagi, Keiko ; Wada, Housei ; Ochiai, Hiroshi ; Miyamoto, Tatsuo ; Kawai, Narudo ; Sasakura, Yasunori ; Matsuura, Shinya ; Okada, Yasushi ; Kawahara, Atsuo ; Hayashi, Shigeo ; Yamamoto, Takashi. / Efficient TALEN construction and evaluation methods for human cell and animal applications. In: Genes to Cells. 2013 ; Vol. 18, No. 4. pp. 315-326.
@article{3115e9f231ab47dbaaf4d7185e584454,
title = "Efficient TALEN construction and evaluation methods for human cell and animal applications",
abstract = "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/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 1 week. 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.",
author = "Tetsushi Sakuma and Sayaka Hosoi and Knut Woltjen and Suzuki, {Ken Ichi} and Keiko Kashiwagi and Housei Wada and Hiroshi Ochiai and Tatsuo Miyamoto and Narudo Kawai and Yasunori Sasakura and Shinya Matsuura and Yasushi Okada and Atsuo Kawahara and Shigeo Hayashi and Takashi Yamamoto",
year = "2013",
month = "4",
doi = "10.1111/gtc.12037",
language = "English",
volume = "18",
pages = "315--326",
journal = "Genes to Cells",
issn = "1356-9597",
publisher = "Wiley-Blackwell",
number = "4",

}

TY - JOUR

T1 - Efficient TALEN construction and evaluation methods for human cell and animal applications

AU - Sakuma, Tetsushi

AU - Hosoi, Sayaka

AU - Woltjen, Knut

AU - Suzuki, Ken Ichi

AU - Kashiwagi, Keiko

AU - Wada, Housei

AU - Ochiai, Hiroshi

AU - Miyamoto, Tatsuo

AU - Kawai, Narudo

AU - Sasakura, Yasunori

AU - Matsuura, Shinya

AU - Okada, Yasushi

AU - Kawahara, Atsuo

AU - Hayashi, Shigeo

AU - Yamamoto, Takashi

PY - 2013/4

Y1 - 2013/4

N2 - 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/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 1 week. 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.

AB - 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/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 1 week. 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.

UR - http://www.scopus.com/inward/record.url?scp=84875618956&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84875618956&partnerID=8YFLogxK

U2 - 10.1111/gtc.12037

DO - 10.1111/gtc.12037

M3 - Article

C2 - 23388034

AN - SCOPUS:84875618956

VL - 18

SP - 315

EP - 326

JO - Genes to Cells

JF - Genes to Cells

SN - 1356-9597

IS - 4

ER -