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Highly Reliable Creation of Floxed Alleles by Electroporating Single-cell Embryos by Monica F. Sentmanat; J. Michael White; Evguenia Kouranova; Xiaoxia Cui is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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## Abstract ## Background Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. ## Results Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). ## Conclusions Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

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Author

Monica F. Sentmanat; J. Michael White; Evguenia Kouranova; Xiaoxia Cui

Publisher

Springer Science and Business Media LLC

Published

2022

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)