The beginning of a new era in genetics started in the late 80s when two great ideas, homologous recombination to change an endogenous gene in embryonic stem cells (ES) cells came together to generate targeted mice with specific mutations in any gene of choice. This method called gene targeting became the gold standard for determining gene function in mammals. This technique can be used to create traditional knock out (KO) models and floxed alleles to allow the development of conditional gene inactivation in a specific time during development or limited to a specific tissue. Gene targeting also allows us to create knock in (KI) and point mutations to alter the function of specific genes. These mouse models have been key to understanding the roles of individual genes in health and disease. This method has produced more than five hundred different mouse models of human disorders, including cardiovascular and neurodegenerative diseases, diabetes and cancer.
Because of the impact of this powerful technology in all areas of biomedicine, Mario Capecchi, Oliver Smithies and Martin Evans were jointly awarded in 2007 the Nobel Prize in Physiology. Drs. Capecchi and Smithies had the vision that homologous recombination could be used to specifically modify genes in mammalian cells. They experimented independently in zygotes to make this work. Dr. Evans had the idea to use cells established from early mouse embryos (ES) to introduce new genetic material into the mouse germline.
The requires several steps that start with the construction of a DNA vector that has DNA sequences that are homologous to the target gene, together with selectable positive and negative markers that allow for the selection of cells that have incorporated the construct exclusively in the targeted gene. To target genes in mice, the DNA construct is either transfected or electroporated into ES cells in culture where it recombines with the endogenous gene of choice. Only the targeted ES cells are able to grow and proliferate in the presence of neomycin, prior to their injection into a mouse blastocyst. Targeted ES cells mix and form a mosaic with existing cells from which the embryo develops in a surrogate mother. Once mosaic mice are sexually mature, the strategy is to mate them with normal (wt) mice to produce both gene targeted (for example KO) and wt offspring.
Single gene knockout experiments have determined the roles of hundreds of genes in embryonic development, adult physiology, aging and disease. At this point more than 16,000 mouse genes have been knocked out either by deleting a gene or removing exons. Ongoing international efforts will develop KO mouse models for all genes and make them available to the public domain in the near future. In the United States, the NIH has funded three Knockout Mouse Project (KOMP2) centers, including one at The Jackson Laboratory, a leader in biomedical and genetic research, to work together on the immense task of producing and phenotyping KO mice. Combined with the international effort coordinated by the International Mouse Phenotyping Consortium (IMPC), a total of 5,000 knockout mice will be produced and phenotyped by 2016. The JAX KOMP team is pleased to contribute their expertise to this international effort to establish a global resource of knockout mice and related databases of gene function.