Highly reliable creation of floxed alleles by electroporating single-cell embryos

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number31
JournalBMC Biology
Volume20
Issue number1
DOIs
StatePublished - Dec 2022

Keywords

  • CRISPR
  • Conditional knockout mice
  • Floxing
  • Functional genomics
  • Gene editing
  • Mouse models

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