History
Two discoveries have been instrumental for the ability to generate knockout mice, the isolation of stem cells and the discovery of homologous recombination. The significance of these findings was demonstrated when Mario Capecchi, Oliver Smithies, and Martin Evans were awarded the 2007 Nobel Prize in Physiology or Medicine for their work in establishing the knockout mouse model (Vogel, 2007). The key step needed for making knockout mice was the isolation of embryonic stem cells (ES cells). The ES cells were isolated from the inner cell mass of a 3.5 days postcoitum mouse blastocyst (Evans and Kaufman, 1981; Martin, 1981). These stem cells could progressively grow in tissue culture and were pluripotent. By injecting the stem cells into a mouse blastocyst, Bradley et al. (1984) were able to generate chimeric mice. The production of chimeric mice proved that the stem cells were able to differentiate into multiple cell lineages and were able to contribute to the development of the mouse embryo. Germline transmission was also achieved by using the pluripotent ES cells. After breeding the chimeric mice, the resulting offspring that were born were clearly derived from the ES cells as seen with the transmission of coat color. Therefore, the introduced stem cells could become established into the germline of the chimeric mice. The isolation of stem cells basically meant that an ES cell clone that was genetically modified in culture could eventually be used to derive a mouse.