Kobayashi Kenichi, Tsukiyama Tomoyuki, Nakaya Masataka, Kageyama Susumu, Tomita Keiji, Murai Ryosuke, Yoshida Tetsuya, Narita Mitsuhiro, Kawauchi Akihiro, Ema Masatsugu
Department of Urology, Shiga University of Medical Science, Japan.
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.
Stem Cell Res. 2019 May;37:101439. doi: 10.1016/j.scr.2019.101439. Epub 2019 Apr 15.
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 Zeocin 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 Zeocin 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.
食蟹猴胚胎干细胞（Cyn ES）可以用与人类胚胎干细胞相似的方式产生。然而，未经训练的研究人员很难将食蟹猴胚胎干细胞维持在未分化状态。为了更易于培养，我们使用CRISPR/Cas9基因组编辑技术构建了一种OCT3/4-P2A-tdTomato-IRES-博来霉素食蟹猴胚胎干细胞系。通过同源重组，将内源性OCT3/4基因座的终止密码子替换为P2A-tdTomato-IRES-博来霉素聚腺苷酸盒。该细胞系使我们能够通过添加博来霉素分离多能干细胞并排除分化细胞，特别是在无饲养层细胞的培养中。
