Targeting multi-cellular organisms.
For many years, biologists' efforts in achieving gene targeting by homologous recombination in multicellular organisms have been hampered by the difficulty in culturing pluripotent stem cells. Recent advances have eliminated this requirement for several animal species. For large mammals, cloning by nuclear transfer has led to the first knockout sheep and pigs. For Drosophila, whole organism gene targeting was accomplished by a method for the generation of linear DNA in vivo.