CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Leading Edge Essay Gene Targeting in Embryonic Stem Cells Scores a Knockout in Stockholm Tak Wah Mak1,* 1Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, and the Departments of Medical Biophysics and Immunology, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada M5G 2C1 *Correspondence: [email protected] DOI 10.1016/j.cell.2007.11.033 The 2007 Nobel Prize in Physiology or Medicine has been awarded to Mario Capecchi, Martin Evans, and Oliver Smithies for developing specific gene modification techniques and mouse embryonic stem cell technology that, when combined, enable the creation of “knockout” mice. Analyses of these mutant animals have revolutionized the elucida- tion of gene functions, and these mice have proved to be valuable models of numerous human diseases. Exactly a century ago, Clarence Cook Martin Evans of Britain’s Cardiff Uni- immune responses are all strikingly Little, a graduate student in William versity, and Oliver Smithies of the Uni- comparable at the physiological level Ernest Castle’s laboratory at Harvard versity of North Carolina at Chapel Hill. in humans and mice. Perhaps this University, clung to the belief that the The citation reads: “For their discover- should not be surprising, as 99% of lowly mouse could one day become a ies of principles for introducing specific the genes in these two species are model in which to study human physi- gene modifications in mice by the use shared. Thus, the use of the mouse ology and disease. Little and Castle of embryonic stem cells.” This year’s as a model for studying human devel- realized that, to achieve this goal, Nobel Prize nicely complements last opment and disease is an approach they had to develop mouse strains year’s award to Andrew Fire of Stanford that can be readily justified. that were more genetically homoge- University and Craig Mello of the Uni- neous. Backed by Castle’s expertise versity of Massachusetts at Worcester, The Early Days of Mouse Genetics as a respected authority on mamma- who jointly received the 2006 Nobel The early pioneers of mouse genet- lian Mendelian genetics, Little com- Prize for Physiology or Medicine for ics were well aware of the possibili- menced interbreeding wild mice in their discovery of microRNAs. The prin- ties of using rodents to learn more 1909. He hoped to obtain animals with ciples by which microRNAs function about human biology and genetics. a better defined genetic background have been exploited to develop inter- Little, Leonell Strong, E. Carlton Mac- that would simplify laboratory studies ference RNAs that permit the silencing Dowell, and others spent almost two of mammalian traits. The success of of gene functions at will and with rela- decades systematically intercross- this program marked the beginning of tive ease. ing mice captured from the wild and inbred mouse genetics. generating scores of inbred strains. A This year, we celebrate a century Why Study Mice? dozen of these lines are still commonly of advances in a subject that has per- Scientists study biology to learn about used in laboratories around the globe, meated every field of physiology and physiology and investigate mammals including the Balb/c, B6, B10, C3H, medicine: animal genetics. We rejoice to learn about human behavior, devel- CBA, and DBA strains. Comparative that the Nobel Assembly at the Karo- opment, and pathophysiology. At the investigations of these multiple lines linska Institute in Sweden has awarded cellular and molecular levels, there of mice derived from a mixture of the 2007 Nobel Prize in Physiology or are significant similarities between forebears from relatively diverse geo- Medicine to three individuals who pio- human cells and those of other multi- graphic locations have allowed scien- neered techniques of gene targeting cellular or even unicellular organisms. tists to probe the extent of mammalian in murine embryonic stem (ES) cells. However, at the organismal level, genetic diversity. Many of the mouse The work of these researchers revolu- humans share extensive physiologi- strains created by these researchers, tionized the study of mouse genetics cal characteristics only with other pri- as well as other rodent mutants, were and has made it possible for scientists mates. Nevertheless, many features eventually consolidated at the Jack- around the world to generate geneti- of human development and biology son Laboratory in Maine, which Little cally defined mouse mutants for the are closely analogous to those of founded in 1929. The Jackson Labora- study of functions of individual genes. fast-breeding and easily maintained tory became and remains to this day The 2007 Nobel Prize winners are Mario rodents. Embryonic development, one of the meccas of rodent genetics, Capecchi of the University of Utah, organogenesis, hematopoiesis, and devoted to the unearthing and housing Cell 131, December 14, 2007 ©2007 Elsevier Inc. 1027 of interesting substrains and mutants. mice in question were genetically became a priority. Solving this difficulty Back in the 1930s, investigations of more closely related than was ideal. was never as dire in other species as the Jackson Laboratory’s collection Underneath their ostensible diversity, it was in mammalian cells, for obvious of mutants yielded major advances in these rodents actually arose from a reasons. Unicellular organisms such our understanding of many aspects relatively small number of ancestors as bacteria and yeast, and even multi- of mammalian bodily processes. For derived from a limited number of orig- cellular species like the fruit fly Droso- example, Peter Gorer and George inal sources. Even with the benefit of phila melanogaster and the nematode Davis Snell, another student of Castle, modern techniques of irradiation and Caenorhabditis elegans, reproduce at joined the Jackson Laboratory and chemical mutagenesis, the process of a much faster rate than mammals and devoted 25 years almost exclusively obtaining genetic variants via breed- are much more amenable to mutagen- to studies of mouse histocompatibility ing programs remains lengthy, costly, esis. Mammalian geneticists dreamt genes. In the course of these stud- and labor intensive. of refining the techniques of “forward ies, Snell discovered the H-2 complex genetics” used to generate mutations (containing the MHC genes) that gov- Molecular Biology Revolutionizes in flies and worms and applying them erns transplant rejection and immune Mouse Genetics to mammalian cells. A technology that responses. For this work, Snell shared With the dawn of the molecular biology went a long way to satisfying the desire the 1980 Nobel Prize in Physiology or era in the mid-1970s, it became pos- of biologists to study the functions Medicine with Jean Dausset and Baruj sible to identify the molecular bases of specific genes in whole animals Benacerraf. of the physiological and pathophysi- was the independent development Little’s trailblazing efforts were fol- ological variations observed in differ- of transgenic mice in the early 1980s lowed by the heroic labors of many ent mouse strains and their mutants. by Rudolf Jaenisch of the Massachu- who undertook the arduous task of However, early attempts to delineate setts Institute of Technology, Frank creating and characterizing mouse these mutations at the molecular Ruddle of Yale University, and Ralph models of human diseases. Although level required an enormous struggle Brinster of the University of Pennsyl- the initial objective was to study tum- and consumed years of demanding vania and Richard Palmiter of the Uni- origenesis in mice, the intercrossing experimentation. Even with today’s versity of Washington (among others) of millions of animals over several technology, this type of “forward (Palmiter and Brinster, 1985). These decades also produced rare exam- genetics” approach requires consid- mutant animals allowed researchers ples of mice exhibiting symptoms of erable effort to pinpoint the molecular to assess a gene’s function by exam- anemia, immunodeficiency, fragile X changes occurring in randomly gen- ining the effects of its overexpression syndrome, Alzheimer’s dementia, or erated mutants. We now know, as a in either a whole animal or a specific obesity (among others). The work of result of two massive DNA sequenc- tissue. Thousands of such mice were many dedicated individuals contrib- ing projects carried out by Fernando produced and studied and the appli- uted to this cause, including the pio- Pardo-Manuel de Villena at the Uni- cation of this technology is still preva- neering research of Elizabeth Russell, versity of North Carolina and his col- lent today. Nevertheless, despite the Sheldon Bernstein, and Jane Baker laborators at the Jackson Laboratory, usefulness of transgenic mice, mam- on mouse anemia; the landmark as well as by David Cox of Perlegen malian geneticists still sought a means experiments of Douglas Coleman on Sciences Inc., that the genomes of of mutating a gene and observing the obesity; and Donald Bailey’s ground- the commonly used laboratory strains results of its loss of function in a whole breaking development of recombi- and several lines of wild mice differ by animal. Thus, the goal was to gener- nant inbred strains that facilitate gene only a few million base pairs (Calla- ate somatic cell mutants or, better still, mapping. William and Lee Russell of way, 2007). This amount of variation, gene-targeted mice bearing specific the Oak Ridge National Laboratory which is unevenly distributed in the alterations. The trick was to find a way in Tennessee, as well as Mary Lyon mouse genome, is considerably less to incorporate a defined mutation into and Bruce Cattanach of the Atomic than expected. The creation of addi- the genome of a mammalian embryo Energy Research Establishment in tional rodent strains from stocks of a that could then develop into a whole Hartwell in England, led research more diverse genetic background are animal displaying the effects of that teams that formalized irradiation and currently in progress.