Abstract
Xenopus tropicalis is a versatile model organism for studying basic biology such as developmental biology and cell biology, and for biomedical research on human diseases. Current genome editing techniques enable researchers to easily perform gene targeting in various animals. Among them, gene knockout using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system has recently become an indispensable strategy for loss-of-function analysis in vivo. Because of its ease of use, time, and cost efficiencies, CRISPR-Cas has also been applied to X. tropicalis where the gene disruption is highly efficient. In this chapter, we introduce a simple CRISPR-Cas system protocol for gene disruption in X. tropicalis. Based on our protocol, researchers can generate knock-out phenotypes within the shortest of timeframes, a week, and analyze genes of interest in founder generation.
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Acknowledgments
We are grateful to Drs. Akihiko Kashiwagi and Keiko Kashiwagi (Hiroshima University) for technical advice on animal husbandry and to Ms. Mitsuki Shigeta and Miyuki Suzuki for experimental support. X. tropicalis (Golden strain) was provided by the Institute for Amphibian Biology (Hiroshima University) through the National Bio-Resource Project, Japan Agency for Medical Research and Development, Japan. This work was supported by the Japan Society for the promotion of Science KAKENHI Grant Number 15K06802 [Grant-in-Aid for Scientific Research (C)], The Narishige Zoological Science Award and The Naito Foundation to K.T.S. and JSPS KAKENHI Grant Number 15J05833 [Grant-in-Aid for JSPS Fellows] to Y.S.
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Sakane, Y., Suzuki, Ki.T., Yamamoto, T. (2017). A Simple Protocol for Loss-of-Function Analysis in Xenopus tropicalis Founders Using the CRISPR-Cas System. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 1630. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7128-2_16
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DOI: https://doi.org/10.1007/978-1-4939-7128-2_16
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