Efficient Selection of Knocked-In Pluripotent Stem Cells Using Dual Cassettes of Cellular Suicide System (original) (raw)
45 Pages Posted: 6 Dec 2022 Publication Status: Published
Koji Nakade
RIKEN BioResource Research Centre - Gene Engineering Division
Satomi Tsukamoto
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Kenichi Nakashima
RIKEN BioResource Research Centre - Gene Engineering Division
Yuri An
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Iori Sato
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Jingyue Li
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Yasuko Hemmi
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Yoshihiro Miwa
RIKEN BioResource Research Centre - Gene Engineering Division
Yohei Hayashi
RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team
Abstract
Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we have developed the “double-tk donor vector system” in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knock-in hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B Clustered Histone 21), and ISL1. These results indicate that this cellular suicide system based on double-tk donor vectors enabled the efficient selection of knocked-in carrying reporter proteins.
Note:
Funding Information: This research was supported in part by grants from AMED (21ae0201004s0504) to Y.M., from JST (JPMJPF2017) to Y.M., and from JSPS KAKENHI Grant-in-Aid for Young Scientists (A) (17H05063) to Y.H., and from Scientific Research (C) (21K05994) to Y.M.
Declaration of Interests: The authors declare no competing interests.
Keywords: human induced pluripotent stem cells, Genome editing, knock-in, thymidine kinase of herpes simplex virus, homology-directed repair, fluorescent proteins, donor vectors
Suggested Citation: Suggested Citation
Nakade, Koji and Tsukamoto, Satomi and Nakashima, Kenichi and An, Yuri and Sato, Iori and Li, Jingyue and Hemmi, Yasuko and Miwa, Yoshihiro and Hayashi, Yohei, Efficient Selection of Knocked-In Pluripotent Stem Cells Using Dual Cassettes of Cellular Suicide System. Available at SSRN: https://ssrn.com/abstract=4290057 or http://dx.doi.org/10.2139/ssrn.4290057
This version of the paper has not been formally peer reviewed.