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Efficient Generation of Gene-Modified Mice by Haploid Embryonic Stem Cell-Mediated Semi-cloned Technology

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In Vitro Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1498))

Abstract

Haploid embryonic stem cells can be derived from androgenetic embryos produced by injection of sperm into enucleated oocytes or by removal of the female pronucleus from zygotes. These cells, termed AG-haESCs, can be used in place of sperm to produce the so-called semi-cloned (SC) mice. Importantly, AG-haESCs carrying H19-DMR and IG-DMR knockouts (DKO-AG-haESCs) can efficiently and stably support the generation of SC mice via intracytoplasmic AG-haESCs injection (ICAHCI), which provides a new route to obtain genetically modified mice. In this chapter, we describe the procedures for AG-haESCs culturing, enrichment of haploid cells by FACS, genomic manipulation in DKO-AG-haESCs by CRISPR/Cas9 and generation of live SC mice with gene-modified DKO-AG-haESCs.

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References

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Acknowledgments

This study was supported by grants from the Ministry of Science and Technology of China (2014CB964803 and 2015AA020307), the National Natural Science Foundation of China (91319310, 31225017, and 31530048), the Chinese Academy of Sciences (XDA01010403 and XDB19010203), and the Shanghai Municipal Commission for Science and Technology (12JC1409600 and 13XD1404000).

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Authors and Affiliations

  1. Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Cuiqing Zhong & Jinsong Li

  2. Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Cuiqing Zhong & Jinsong Li

Authors
  1. Cuiqing Zhong
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  2. Jinsong Li
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Corresponding author

Correspondence to Jinsong Li .

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Editors and Affiliations

  1. Coskata, Inc., Warrenville, Illinois, USA

    Andrew Reeves

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Zhong, C., Li, J. (2017). Efficient Generation of Gene-Modified Mice by Haploid Embryonic Stem Cell-Mediated Semi-cloned Technology. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6472-7_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6470-3

  • Online ISBN: 978-1-4939-6472-7

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