ABSTRACT
The introduction of exogenous DNA into mammalian cells has proven to be a very useful approach to study the mechanisms controlling gene regulation. In principle, the repair of genetic defects can be achieved by introducing one or more copies of the gene of interest into the genome by random integration. Successive trypsinization of the growing inner cell mass cells and plating on new feeder cells yields a homogenous population of embryonic stem cells. Many strategies have therefore been developed to enhance the homologous recombination frequency in mammalian cells, especially embryonic stem (ES) cells. The generation of loss-of-function mutants by homologous recombination in ES cells uses the insertion of the neomycin gene or other selectable marker in the targeting construct. Gene families can be knocked out one by one, and single, double and eventually triple mutants can be analysed to elucidate the role of the different genes and to rule out redundancy effects.
