Abstract
Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice.
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Acknowledgment
This work was supported by grants from the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from Agency for Medical Research and Development (AMED) under Grant Number JP22ama121049, and the Practical Research Project for Rare/Intractable Diseases from AMED.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Kobayashi, R., Horii, T., Hatada, I. (2023). Efficient Detection of Flox Mice Using In Vitro Cre Recombination. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 2637. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3016-7_12
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DOI: https://doi.org/10.1007/978-1-0716-3016-7_12
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