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Gene Targeting in Embryonic Stem Cells Scores a Knockout in Stockholm

Tak Wah Mak1,* 1Campbell Family Institute for Breast Research, Ontario Cancer Institute, and the Departments of Medical Biophysics and , , 620 University Avenue, Toronto, Ontario, Canada M5G 2C1 *Correspondence: [email protected] DOI 10.1016/j.cell.2007.11.033

The 2007 in or has been awarded to , , and for developing specific modification techniques and mouse embryonic 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 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 of Stanford that can be readily justified. that were more genetically homoge- University and of the Uni- neous. Backed by Castle’s expertise versity of Massachusetts at Worcester, The Early Days of Mouse 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 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 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- 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 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 , 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 , 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 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 (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 to study the functions Medicine with 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 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 , 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- , 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 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 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. mutation. other mutagenesis techniques that While the enterprise of mouse made it easier to study basic ques- genetics was steadily advancing from From Multipotent Cell to Mutant tions in mouse genetics. However, the 1930s to the 1980s, the study of Animal despite these prodigious efforts, the genes themselves was undergo- The development of a whole animal elucidation of the genetic causes ing a revolution. By the late 1970s, the from an embryo requires that the earli- of physiological phenomena such techniques of molecular biology were est embryonic cells be multipotent, that as cancer and obesity proved to be starting to deliver significant numbers is, have the capacity to generate every exceedingly strenuous. One impedi- of genes to biologists for study, and cell type needed in the body. The multi- ment to more rapid progress in these the need for a quick and easy means potency of mammalian cells has fasci- studies was the discovery that the of generating defined mouse mutants nated embryologists and biologists for

1028 Cell 131, December 14, 2007 ©2007 Elsevier Inc. decades. In 1961, Ernest A. McCulloch and James E. Till at the Ontario Can- cer Institute in Toronto discovered that a single bone marrow precursor cell capable of forming a colony in an irra- diated recipient mouse could give rise to multiple lineages of hematopoietic cells (Till and McCulloch, 1961). This result clearly demonstrated that at least some mammalian progenitor cells had the ability to differentiate into cell types of different lineages and suggested that it might be possible to manipulate the course of organogenesis. These exper- iments set the stage for the very ambi- tious dream (at the time) of regenerat- ing an entire animal by injecting a small number of multipotent cells into an early embryo. The ability to propagate in culture, or isolate from animals, pre- cursor cells having the capacity to give rise to an entire mammal would allow researchers to manipulate the genetics of a whole animal. The first step in this direction came from the work of Roy Stevens at the Jackson Laboratory, G. Barry Pierce of the University of Colo- rado, and Beatrice Mintz at the Fox Chase Institute in Philadelphia (among others). These researchers reported on studies in which teratocarcinoma cell lines were induced to differentiate into cells of various tissue types, yielding invaluable insights into the plasticity of multiple lineage commitment (Andrews, 2002). However, teratocarcinoma cells could not be used to regenerate an entire animal because these cells are intrinsically tumorigenic. The break- through came in 1981 when Evans and his colleagues and Gail Martin’s laboratory independently succeeded in developing ES cell lines (Evans and Kaufman, 1981; Martin, 1981). These cells, which were derived from an early murine , grew indefinitely in Figure 1. How to Make a The first step in generating a knockout mouse is the construction of the targeting vector. The tissue culture and retained their multi- targeting vector generally contains a copy of the genomic murine gene disrupted by the inser- potency as long as they were cultured tion of a positive selection marker such as neomycin resistance (Neo). A negative drug selection on feeder layers. When injected into a gene is also often included. The targeting vector is then introduced into murine ES cells, usually by electroporation. Successive rounds of drug selection allow the isolation of ES cells that are new blastocyst, these ES cells contrib- homologous recombinants. Confirmation of the desired gene disruption is achieved by PCR and uted to the developing murine embryo, Southern blotting of the ES cell DNA. ES cells heterozygous for the gene disruption are then resulting in the creation of a genetic injected into mouse to generate embryos that are transferred to pseudopregnant mosaic. The chimeric embryos were female mice. Chimeric progeny are born that show incorporation of the targeted gene into either somatic cells or the . Chimeric mice with germ cells bearing the targeted mutation are then brought to term by implantation in bred with wild-type mice to generate progeny heterozygous for the disruption. Intercrossing of a pseudopregnant female mouse (see these heterozygotes produces F1 progeny, one-quarter of which should be mutants homozygous Figure 1). This seminal work provided for the targeted mutation: the classic knockout mouse. (Figure adapted from The Immune Re- sponse: Basic and Clinical Principles by Tak W. Mak and Mary E. Saunders, 2006, published by researchers with the opportunity to Elsevier Academic Press, London, UK). manipulate the genetics of a mammal

Cell 131, December 14, 2007 ©2007 Elsevier Inc. 1029 at the embryonic stage. The Evans lab exchange. However, it was not known lying this process. Today, a veritable provided further proof of this principle whether this type of homologous cottage industry of hundreds of labo- by introducing the DNA of a retroviral recombination could be induced in ratories worldwide has produced an provirus into ES cells and documenting mammalian cells through the intro- estimated 10,000 different types of the transmission of this foreign DNA duction of naked foreign , or genetically engineered mice. Further- through multiple generations of mice whether such introduction would be more, an international consortium has (Robertson et al., 1986). efficient enough to permit the creation recently been formed with an eye to Despite these triumphs, technical of specific genetic changes. The lab- mutating all protein-encoding genes challenges remained in consistently oratories of Capecchi and Smithies in the mouse genome using this tech- sustaining ES cells in their multipo- were the first to show that homolo- nology. At this point, the bottleneck tent state in culture. In 1988, Aus- gous recombination between plas- to progressing faster in defining gene tin Smith and John Heath at Oxford mid DNAs could be detected when functions lies in analyzing the phe- University and their collaborators at the plasmids were introduced into notypes of these animals rather than the Genetics Institute in Boston, as mammalian cells. These investigators creating them. well as Nicholas Gough of the Euro- also subsequently demonstrated that As the gene-targeting juggernaut pean Molecular Biology Laboratory in specific mutations could be incor- gathered momentum in the mid- Heidelberg, discovered that inclusion porated into mammalian DNA via 1990s, results were accumulating in the culture medium of the growth the introduction of a plasmid-based that had far-reaching consequences. factor leukemia inhibitory factor (LIF) vector bearing foreign DNA. Surpris- It was soon discovered that null gene- allowed ES cells to robustly retain ingly, a reasonably high degree of targeted mutations, which disrupt their multipotency. With this diffi- recombination was observed even gene function in every tissue of an culty conquered, ES cell technology when the vector and the intended animal, often precluded the study of a became well defined and reproduc- genomic target sequence showed gene’s function in adult tissues. Thus, ible. The ability to reliably maintain ES only a few kilobases of homology. In other genetic modifications in rodents, cells in culture has allowed investiga- 1987, Capecchi’s group succeeded including deletions, insertions, inver- tors to work out the principles and in mutating the HPRT gene in ES sions, translocations, and point muta- mechanisms of stem cell self-renewal cells by gene targeting (Thomas and tions, were devised to study the rela- and to identify critical regulators of Capecchi, 1987). Independently, the tionship between a gene’s structure tissue/cell differentiation. However, introduction of a vector bearing the and its function. A further refinement back in the 1980s, the more important wild-type HPRT gene sequence was of this approach was to conditionally observation was that ES cells offered used to target and functionally cor- delete or mutate genetic loci in an an unprecedented opportunity to rect a mutated HPRT gene in an ES inducible fashion, such that specific view the physiological consequences cell (Doetschman et al., 1987). These genetic alterations could be turned of specific genetic changes such as efforts formally proved that homolo- “off” or “on” in a temporal or spatial point mutations, insertions, and dele- gous recombination could be used manner. The technology to create tions. The next hurdle to overcome to modify the mammalian genome in these conditional mutations arose was finding a method of introducing a predetermined manner, paving the from an ingenious application of the such genetic alterations into a mam- way for the creation of gene-targeted bacteriophage enzyme Cre recom- malian genome. “knockout” mice. binase by the laboratory of Klaus Rajewsky, then at the University of : A Universe of Knockout Mice Koln (Gu et al., 1994). By placing Cre Mutations to Order The combination of the technologies recombinase under the control of a It had long been known that bac- of ES cell manipulation and homolo- tissue-specific or stage-specific pro- teria and yeast could repair dam- gous recombination provided the moter and flanking the gene to be tar- age to their DNA through a process powerful “reverse genetics” approach geted with the loxP sites recognized known as homologous recombina- that had been long sought to gener- by Cre, researchers could choose tion. Homologous recombination is ate genetically defined rodents for the the timing and location of deletion of a natural route by which a stretch study of mammalian physiology and the gene of interest. These and other of mutated DNA can be exchanged pathophysiology. Dozens of labora- variations on gene-targeting tech- with a functional copy of this region tories dived into the arena and dis- niques have led to the establishment of the genome if there is extensive covered that, despite its complexity, of an estimated 500 mouse models of nucleotide similarity between the two the technology was relatively easy human diseases ranging from cancer, DNA sequences. By the early 1980s, to master. Within a decade, over one , and cardiovascular disease studies of the somatic recombination thousand mutated mice were gener- to neurological ailments. In addition of gene segments of the B and T cell ated. In addition, the ability to manip- to the basic research conducted on receptor genes had made it clear that ulate homologous recombination in these mutants, pharmaceutical com- mammalian cells could also carry out mammalian cells energized the study panies frequently use them to aid in this mechanism of DNA sequence of the molecular mechanisms under- drug discovery and testing.

1030 Cell 131, December 14, 2007 ©2007 Elsevier Inc. From Knockout Mice to Healthier less tumorigenic than teratocarci- to greatly benefit human health. Many Humans noma cells. Just three weeks ago, believe that the 21st century will be Like all outstanding work, the dis- the laboratories of the era in which studies of human covery of mouse ES cells and gene and James Thomson of the Genome genetics lead directly to regenerative targeting has had an impact much Center of Wisconsin reported using medicine. If so, Little’s conviction will greater than what might have been a similar transduction approach to have been validated beyond his fond- expected. Many researchers muse generate induced pluripotent stem est imaginings. that parallel studies of human ES cells from human somatic cells (Taka- cells could perhaps move us closer hashi et al., 2007; Yu et al., 2007). Acknowledgments to understanding true human traits. These human ES-like cells express Such understanding could some day ES cell-surface markers, have normal I thank B.G. Neel, J. Rossant, and M. Saun- ders for helpful discussions. spur the use of this technology for karyotypes, express telomerase, and the purposes of regenerative medi- are capable of differentiating into cell References cine. However, such research would types of all three germ layers. This entail the manipulation of human ES exciting scientific advance means Andrews, P.W. (2002). Philos. Trans. R. Soc. cells, a procedure that is considered that it may soon be possible to pro- Lond. B Biol. Sci. 357, 405–417. unethical in many countries today. It vide patients with multipotent stem Callaway, E. (2007). 448, 516–517. is unlikely that this controversy will cells tailored for a given therapeutic evaporate in the near future because purpose. Because these ES-like cells Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton, D.W., Thompson, S., gene-targeting techniques currently would have been generated from the and Smithies, O. (1987). Nature 330, 576–578. rely on ES cells, and human ES cells patient’s own fibroblasts, the chance Evans, M.J., and Kaufman, M.H. (1981). Na- can only be derived from human of the patient’s immune system ture 292, 154–156. embryos. However, several groups of rejecting them upon transplantation investigators made a discovery last would be minimal. Even more satisfy- Gu, H., Marth, J.D., Orban, P.C., Mossmann, H., and Rajewsky, K. (1994). Science 265, year that promises to provide a sur- ing may be the prospect that, using 103–106. rogate approach to studying human the homologous recombination pro- embryonic development and physiol- cedure established by Capecchi and Martin, G.R. (1981). Proc. Natl. Acad. Sci. USA 78, 7634–7638. ogy without having to directly inves- Smithies, ill effects from aberrations tigate human ES cells. Working in present in the germline of patients Palmiter, R.D., and Brinster, R.L. (1985). Cell mice, teams led by Shinya Yamanaka might be treated using tissue-specific 41, 343–345. of , Rudolph Jae- ES-like cells. These possibilities show Robertson, E., Bradley, A., Kuehn, M., and nisch at the Massachusetts Institute that the work of Evans, Capecchi, and Evan, M. (1986). Nature 323, 445–448. of Technology, and Kathrin Plath of Smithies has boundless implications Rossant, J. (2007). Nature 448, 260–262. the University of California at Los for the scientific, medical, and ethical Angeles and Konrad Hochedlinger of arenas. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. the Massachusetts General Hospi- As we move into the second cen- (2007). Cell 131, 1–12. tal have found that a small percent- tury of studying mouse genetics, we Thomas, K.R., and Capecchi, M.R. (1987). Cell age of murine skin fibroblasts can be eagerly await the next great discov- 51, 503–512. transduced to revert to multipotent eries in murine and human genetic stem cells by the introduction of only manipulation that will further advance Till, J.E., and McCulloch, E.A. (1961). Radiat. Res. 14, 213–222. four transcription factors (Rossant, medical science. By awarding the 2007). These “reprogrammed” cells, 2007 Nobel Prize to the discoverers of Yu, J., Vodyanik, M.A., Smuga-Otto, K., Anto- which are termed “induced pluripo- knockout mice, the international com- siewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et tent stem cells,” are quite similar in munity has vindicated Little’s original al. (2007). Science. Published online Novem- their properties to ES cells but are faith in the power of small creatures ber 20, 2007. 10.1126/science.1151526.

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