Efficient Detection of Flox Mice Using In Vitro Cre Recombination

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.