Generation of Floxed Mice by Sequential Electroporation

Methods Mol Biol. 2023:2637:135-147. doi: 10.1007/978-1-0716-3016-7_11.

Abstract

Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.

Keywords: CRISPR-Cas9; Cre-loxP; Electroporation; Flox; Knock-in; Mouse; loxP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Electroporation Therapies*
  • Electroporation*
  • Integrases / genetics
  • Mice
  • Mice, Knockout
  • Mice, Transgenic

Substances

  • Integrases