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Updated: Aug 10, 2025
Generation of Mice Derived from Induced Pluripotent Stem Cells
Published on: November 29, 2012
Generation of Floxed Mice by Sequential Electroporation
Takuro Horii1, Ryosuke Kobayashi2, Izuho Hatada3,4
1Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan. horii@gunma-u.ac.jp.
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.
Insights
This study introduces a sequential electroporation method to improve the efficiency of generating conditional knockout mice. The new technique enhances the floxing rate, leading to more offspring with targeted gene modifications.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Conditional knockout mice are crucial for studying gene function using the Cre-loxP system.
- Current CRISPR-Cas9 methods for simultaneous loxP insertion can cause large chromosomal deletions and low floxing efficiency.
Purpose of the Study:
- To develop an improved method for generating conditional knockout mice with higher floxing efficiency.
- To overcome the limitations of simultaneous loxP insertion using CRISPR-Cas9.
Main Methods:
- Developed a sequential electroporation technique to introduce loxP sequences.
- Applied electroporation at one- and two-cell embryonic stages for sequential insertion.
- Utilized CRISPR-Cas9 technology in conjunction with the sequential approach.
Main Results:
- The sequential electroporation method significantly improved floxing efficiency compared to simultaneous methods.
- Achieved a higher yield of offspring carrying floxed alleles.
- Reduced undesirable large chromosomal deletions associated with simultaneous insertion.
Conclusions:
- Sequential introduction of loxP sites via electroporation is a more efficient strategy for generating conditional knockout mice.
- This method offers a valuable advancement for genetic research and the analysis of gene function.
