Abstract
Following completion of the genome sequences of Xenopus tropicalis and X. laevis, gene targeting techniques have become increasingly important for the further development of Xenopus research in the life sciences. Gene knockout using programmable nucleases, such as TALEN and CRISPR/Cas9, has reached a level whereby we can readily and routinely perform loss-of-function analysis of genes of interest in these species. However, there is still room for improvement in gene knock-in techniques owing to some technical problems. To overcome these problems, several knock-in techniques have been developed. Among them, we introduce in this chapter a simple knock-in system mediated by microhomology mediated end joining repair. This protocol allows us to produce knock-in animals for in vivo tagging, promoter/enhancer traps, and transgenesis in both of these Xenopus species.
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Acknowledgments
This work was supported by JSPS KAKENHI Grant Number 15 K06802 [Grant-in-Aid for Scientific Research (C)] to K.T.S. and 15 J05833 [Grant-in-Aid for JSPS Fellows] to Y.S, NBRP Fundamental Technologies Upgrading Program in AMED to K.T.S. and T.Y., The Narishige Zoological Science Award and The Naito Foundation to K.T.S. We are grateful to Drs. Akihiko Kashiwagi and Keiko Kashiwagi and the National Bio-Resource Project of X. tropicalis in the Amphibian Research Center (Hiroshima University) for providing X. tropicalis, Golden strain. We also thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
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Suzuki, Ki.T., Sakane, Y., Suzuki, M., Yamamoto, T. (2018). A Simple Knock-In System for Xenopus via Microhomology Mediated End Joining Repair. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_7
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DOI: https://doi.org/10.1007/978-1-4939-8784-9_7
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