1. Highly reliable creation of floxed alleles by electroporating single-cell embryos.
- Author
-
Sentmanat, Monica F., White, J. Michael, Kouranova, Evguenia, and Cui, Xiaoxia
- Subjects
- *
RIBOSOMES , *ALLELES , *EMBRYOS , *KNOCKOUT mice , *LABORATORY mice , *GENOME editing - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF