An instrumental tool that we use to ascertain the function of developmental genes is targeted mutagenesis. This technology allows us to customize mutations for any developmental gene to assess biological function in the context of the whole animal. Targeted mutagenesis arose from two breakthroughs in the mid eighties:
When gene targeting is applied to ES cells, genetic mutations that are engineered into the ES cell genome can be transferred to the whole animal through the germline. In the first generation, a chimeric animal is bred with an appropriate coat colour mate to identify transmission of the ES cell genome in the second generation. Because the original ES cell line is heterozygous, only 50% of these progeny will carry the mutations. These heterozygous animals have to be bred to generate homozygous mutants.
The original and still the most common experiment is the knockout, in which a selectable marker gene is placed into coding sequence to effectively disrupt the gene. This selectable marker aids in the selection of ES cells that have integrated the DNA. Because random integration occurs much more frequently than homologous integration, additional negative selectable markers are placed at the end of the targeting construct, which kill the ES cell if integrated randomly.