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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Yao X, Wang X, Hu X et al (2017) Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res 27:801–814
Yen ST, Zhang M, Deng JM et al (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393:3–9
Dow LE (2015) Modeling disease in vivo with CRISPR/Cas9. Trends Mol Med 21:609–621
Skarnes WC, Rosen B, West AP et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–344
Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109
Yang H, Wang H, Shivalila CS et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/cas-mediated genome engineering. Cell 154:1370–1379
Aida T, Nakade S, Sakuma T et al (2016) Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. BMC Genomics 17:979
Okamoto S, Amaishi Y, Maki I et al (2019) Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Sci Rep 9:4811
Quadros RM, Miura H, Harms DW et al (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18:92
Horii T, Morita S, Kimura M et al (2017) Efficient generation of conditional knockout mice via sequential introduction of lox sites. Sci Rep 7:7891
Horii T, Kobayashi R, Kimura M et al (2020) Calcium-free and cytochalasin B treatment inhibits blastomere fusion in 2-cell stage embryos for the generation of floxed mice via sequential electroporation. Cell 9:1088
Kuno A, Ikeda Y, Ayabe S et al (2022) DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes. PLoS Biol 20:e3001507
Zhu B, Cai G, Hall EO et al (2007) In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43:354–359
Marsischky G, LaBaer J (2004) Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 14:2020–2028
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3016-7_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3015-0
Online ISBN: 978-1-0716-3016-7
eBook Packages: Springer Protocols