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Cell Rep. 2017 Aug 29;20(9):2227-2237. doi: 10.1016/j.celrep.2017.07.081.

Generation of Mouse Haploid Somatic Cells by Small Molecules for Genome-wide Genetic Screening.

Author information

1
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
2
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
3
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; School of Life Sciences, University of Science and Technology of China, Hefei 230026, China.
4
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, Northeast Agricultural University, Harbin 150030, China.
5
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: zhouqi@ioz.ac.cn.

Abstract

The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.

KEYWORDS:

genetic screening; genome-wide; haploid cells; haploidy; stem cells

PMID:
28854370
DOI:
10.1016/j.celrep.2017.07.081
[Indexed for MEDLINE]
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