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Dissecting the Role of the Ccr4–Not Deadenylase Complex in Pluripotency and Differentiation

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Deadenylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2723))

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Abstract

In this chapter, we describe methods and assays to examine the role of the Ccr4–Not mRNA deadenylase complex in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. We present the following procedures: sgRNA design and cloning, the culture of mouse ESCs, transfection and colony picking, genotyping, generation of Cnot3 conditional deletion ESCs, assessment of Cnot3 deletion efficiency, and examination of the impact on ESC maintenance and differentiation. The above approach and protocols can also be readily applied to study other Ccr4–Not subunits in ESC fate regulation, thereby facilitating the systematic dissection of Ccr4–Not function in stem cells and development.

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Acknowledgments

We wish to thank members of the Hu lab for their comments and discussions on this protocol. This work was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences Z01ES102745 (to G.H.).

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Authors and Affiliations

  1. Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

    Xiukun Wang, Qing Chen, Brad Lackford & Guang Hu

Authors
  1. Xiukun Wang
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  2. Qing Chen
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  3. Brad Lackford
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  4. Guang Hu
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Corresponding author

Correspondence to Guang Hu .

Editor information

Editors and Affiliations

  1. RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Eugene Valkov

  2. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA

    Aaron C. Goldstrohm

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© 2024 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply

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Wang, X., Chen, Q., Lackford, B., Hu, G. (2024). Dissecting the Role of the Ccr4–Not Deadenylase Complex in Pluripotency and Differentiation. In: Valkov, E., Goldstrohm, A.C. (eds) Deadenylation. Methods in Molecular Biology, vol 2723. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3481-3_8

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  • DOI: https://doi.org/10.1007/978-1-0716-3481-3_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3480-6

  • Online ISBN: 978-1-0716-3481-3

  • eBook Packages: Springer Protocols

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