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Updated: Oct 4, 2025
Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos
Published on: December 25, 2015
Highly reliable creation of floxed alleles by electroporating single-cell embryos
Monica F Sentmanat1, J Michael White2, Evguenia Kouranova1
1Genome Engineering & Stem Cell Center (GESC@MGI), Department of Genetics, Washington University in St. Louis School of Medicine, 660 S. Euclid Ave, St. Louis, MO, 63110, USA.
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.
