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Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi

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RNAi and Small Regulatory RNAs in Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1622))

Abstract

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

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Acknowledgments

We thank the laboratory members of Organogenesis and Neurogenesis team, and In Vitro Histogenesis team for helpful discussions. We also thank A. Miyawaki for tdKeima cDNA and T. Nakamura and T. Takahito for important advice for the basics of genome editing.

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Authors and Affiliations

  1. Laboratory for In Vitro Histogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo, Kobe, Hyogo, 650-0047, Japan

    Nozomu Takata Ph.D. & Eriko Sakakura M.Eng.

  2. Center for Vascular and Developmental Biology, Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, 60611, Illinois, USA

    Nozomu Takata Ph.D.

  3. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan

    Tetsushi Sakuma Ph.D. & Takashi Yamamoto Ph.D.

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  1. Nozomu Takata Ph.D.
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  2. Eriko Sakakura M.Eng.
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  3. Tetsushi Sakuma Ph.D.
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  4. Takashi Yamamoto Ph.D.
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Correspondence to Nozomu Takata Ph.D. .

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  1. Department of Biology, East Carolina University, Greenville, North Carolina, USA

    Baohong Zhang

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Takata, N., Sakakura, E., Sakuma, T., Yamamoto, T. (2017). Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7108-4_19

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