Generation of an OCT3/4 reporter cynomolgus monkey ES cell line using CRISPR/Cas9

Kenichi Kobayashi; Tomoyuki Tsukiyama; Masataka Nakaya; Susumu Kageyama; Keiji Tomita; Ryosuke Murai; Tetsuya Yoshida; Mitsuhiro Narita; Akihiro Kawauchi; Masatsugu Ema

Stem Cell Research (May 2019)

Generation of an OCT3/4 reporter cynomolgus monkey ES cell line using CRISPR/Cas9

Kenichi Kobayashi,
Tomoyuki Tsukiyama,
Masataka Nakaya,
Susumu Kageyama,
Keiji Tomita,
Ryosuke Murai,
Tetsuya Yoshida,
Mitsuhiro Narita,
Akihiro Kawauchi,
Masatsugu Ema

Affiliations

Kenichi Kobayashi: Department of Urology, Shiga University of Medical Science, Japan
Tomoyuki Tsukiyama: Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Japan; Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Japan; Corresponding authors at: Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan.
Masataka Nakaya: Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Japan
Susumu Kageyama: Department of Urology, Shiga University of Medical Science, Japan
Keiji Tomita: Department of Urology, Shiga University of Medical Science, Japan
Ryosuke Murai: Department of Urology, Shiga University of Medical Science, Japan
Tetsuya Yoshida: Department of Urology, Shiga University of Medical Science, Japan
Mitsuhiro Narita: Department of Urology, Shiga University of Medical Science, Japan
Akihiro Kawauchi: Department of Urology, Shiga University of Medical Science, Japan
Masatsugu Ema: Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Japan; Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Japan; Corresponding authors at: Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan.

Journal volume & issue: Vol. 37

Abstract

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Cynomolgus monkey ES (Cyn ES) cells can be generated in a similar manner as human ES cells. However, Cyn ES cells are difficult to maintain in an undifferentiated state by untrained researchers. For easier culture, we generated an OCT3/4-P2A tdTomato IRES ZeocinR Cyn ES cell line using CRISPR/Cas9 genome editing technology. The stop codon of the endogenous OCT3/4 locus was replaced with the P2A tdTomato IRES ZeocinR pA cassette by homologous recombination. This cell line enables us to isolate pluripotent stem cells and exclude differentiated cells by addition of zeocin, especially for culture without feeder cells.

Published in Stem Cell Research

ISSN: 1873-5061 (Print); 1876-7753 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Biology (General)
Website: https://www.journals.elsevier.com/stem-cell-research

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