Cost-effective generation of mice based on Cre/loxP-system by zygote electroporation of Cre protein
Transgenic Unit of the Czech Centre for Phenogenomics has grown in recent years to offer expanded gene and genome engineering services to researchers by applying the latest cutting-edge technologies such as TALENs, CRISPR/Cas9(Cas12a)-based tools, Easi-CRISPR and others. The introduction of specific mutations by electroporation of zygotes is preferentially used over conventional pronuclear injections and other new transgenic technologies are developing to simplify and accelerate the work. The transgenic team in collaboration with the Laboratory of Structural Biology, IBT AS CR has streamlined a method for generation of mouse models based on Cre/loxP conversion, which has been recently published in Methods.
Cre recombinase mediates recombination between two target sites, named loxP, through the spacer region. The described protocol is applicable to generate knockouts or reporter mice from EUCOMM/KOMP lines and theoretically from any floxed (flanked by loxP) mice. The simplified strategy primarily relies on direct delivery of Cre protein into mouse zygotes via electroporation with nearly 100 percent efficiency and brings several advantages. It simplifies the work, lowers the cost of generating knockout mice and saves time. While a conventional workflow for floxed allele conversion requires a two-step breeding strategy and takes up to six months, the new approach shortens the whole process to one month. Furthermore, Cre protein delivery by electroporation was also shown to be suitable for allelic conversion in primary cells derived from conditional mouse models.
The electroporation-based method allows the delivery of practically any recombinant protein to the cells. Implementing other site-specific recombinases like Flp and Dre will enable the development of more sophisticated mouse models in a relatively short time. Once these recombinases will become available, our facility will be provided with new options for genome engineering and strategies.
Irena Jenickova received the ISTT Best Poster Award for optimization of the technology with her presentation “Zygote Electroporation of Cre and Dre Proteins Enables Efficient Recombination in Mouse Embryos” at the 16th Transgenic Technology Meeting in 2020.
Irena Jenickova, Petr Kasparek, Silvia Petrezselyova, Jan Elias, Jan Prochazka, Jana Kopkanova, Michal Navratil, Cyril Barinka, Radislav Sedlacek: Efficient allele conversion in mouse zygotes and primary cells based on electroporation of Cre protein: Methods. 2020 Jul 24;S1046-2023(20)30114-6. doi: 10.1016/j.ymeth.2020.07.005.