Example of cassette design for homologous recombination

The vector for homologous recombination has been designed such as one of the domain (PDZ) of the gene of interest is interrupted in its coding sequence by the PGK-Neo, marker for the positive selection in ES cells. The TK gene is present outside the cassette to perform a negative selection as well, which greatly facilitates the selection of the ES cells recombinant clones.

The probes (sonde 5 and sonde 3) are designed such as a specific hybridization pattern of a Southern blot can be generated by using the appropriates restriction enzymes (respectively Eco RI and Hind III). The hybridization pattern allows to select recombinant clones or mice from the wild types (5 kb band hybridized for WT and 6.6 kb for KO for the EcoRI/sonde 5 pair; 8.3 kb band for WT and 5.6 band for KO for the HindIII/sonde 3 pair)