Technique of homologous recombination based gene targeting developed in the late 1980s and won the Nobel Prize in Physiology or Medicine in 2007. However, this technique could only performed in mice which embryonic stem (ES) cells could keep in the potential of multifunction in vitro. Therefore gene knockout technology was difficult to be applied in other species of animals for a long time. Since 2008, with the development of the new technologies, such as the new ES cell gene targeting, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clusters of regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), building gene knockout in large and medium animals models which are similar to human in disease research becomes possible. This review describes some new gene knockout technologies in large and medium animal models for recent years.