Gene targeting and site-specific recombination in mouse ES cells

This is a protocol that describes the generation of targeted embryonic stem (ES) cell clones. The targeted cells can be used for generating a mouse either by injection into blastocysts or by morula aggregation. Alternatively, the ES cells can be used for targeting the second allele and thus creating an in-vitro knockout model. In the latter case, the phenotype of the mutation can be analyzed either in the undifferentiated state or after differentiation of the cells into the three germ layers (endoderm, mesoderm, and ectoderm). This protocol describes only a part of the pipeline for generating a conditional knockout mouse. The whole procedure includes (1) design and generation of the targeting construct, (2) generation of targeted ES clones, and (3) generation of the knockout mouse. Detailed protocols for preparing DNA, culturing ES cells, and screening the transfected ES clones for correct targeted events by long-range PCR or Southern blotting can be found elsewhere (see Isolation of Genomic DNA from Mammalian Cells and Analysis of DNA by Southern Blotting). Here, we describe only the protocol used for transfecting the targeting construct into ES cells and for removing antibiotic selection cassettes or other DNA fragments flanked by site-specific recombination target sites using transient transfection of recombinase expression vectors. In addition, we describe a short protocol for screening the clones that underwent complete recombination. A protocol to prepare DNA from 96-, 48-, and 24-well plates is also described.