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Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.

The generation of a new genetically modified mouse strain is a big hurdle to take for many researchers. It is often unclear which steps and decisions have to be made prior to obtaining the desired mouse model. This review aims to help researchers by providing a decision guide that answers the essential questions that need to be asked before generating the most suitable genetically modified mouse line in the most optimal timeframe. The review includes the latest technologies in both the stem cell culture and gene editing tools, particularly CRISPR/Cas9, and provides compatibility guidelines for selecting among the different types of genetic modifications that can be introduced in the mouse genome and the various routes for introducing these modifications into the mouse germline.