PERSPECTIVES in cultured mammalian cells. The second was how to transfer this genetic modification to the mouse germline. Oliver Smithies’ laboratory and mine worked independently on solutions to the first problem. Martin Evan’s laboratory provided us with an approach for a solution RESEARCH PRIZE IN DEVELOPMENTAL BIOLOGY to the second problem. What follows is a description of my laboratory’s contributions to the development of gene targeting in the mouse. It is not meant to be comprehensive; it is rather a more personal description of our contributions to this field. 1977–1980: homologous recombination ESSAY The discoveries that directed my attention to the development of gene targeting began in 1977. At that time, I was exploring whether Gene targeting in mice: functional I could introduce DNA into nuclei of mam- malian cells using extremely small glass analysis of the mammalian genome needles (with tip diameters of less than one micron). Wigler and Axel had just demon- for the twenty-first century strated that mammalian cells deficient in thymidine kinase (tk–) could be transformed into tk+ cells by exposing these cells to a DNA Mario R. Capecchi calcium phosphate co-precipitate containing the herpes virus thymidine kinase (HSV-tk; Abstract | Gene targeting in mouse 20 years after its development, the level of also known as HHV4gp124) gene2. Although embryonic stem cells has become the ‘gold sophistication of genomic manipulations that this was an important advance for the field of standard’ for determining gene function are currently feasible in the mouse through somatic cell genetics, their protocol was not in mammals. Since its inception, this gene targeting can still only be matched in very efficient. With their procedure, incor- technology has revolutionized the study of far simpler organisms, such as bacteria and poration of functional copies of the HSV-tk mammalian biology and human medicine. yeast. gene occurred in approximately one per Here I provide a personal account of the Some investigators have questioned million cells exposed to the DNA calcium work that led to the generation of gene whether such reductionist approaches, phosphate co-precipitate. Using a similar targeting which now lies at the centre of which involve inferring gene function from selection procedure, I asked whether I could functional genomic analysis. the perturbations of a normal phenotype that introduce functional copies of the HSV-tk are induced by the targeted mutations in one gene into mouse tk– fibroblasts using very Gene targeting — creating designed genomic or a small number of genes, have sufficient fine glass needles to inject the DNA directly modifications — has three enormous power to provide significant understanding into their nuclei3. This procedure proved to advantages relative to other procedures for of how truly complex biological phenomena be extremely efficient. One cell in three that introducing mutations into mice. First, the such as higher cognitive functions are medi- received the DNA stably passed the func- investigator chooses which genetic locus ated, particularly in an organism as complex tional HSV-tk gene to its daughters. One to mutate. Second, the technique takes full as the mouse. Frankly, on more gloomy days, does not often observe an almost 106-fold advantage of all the resources provided by I sometimes raise similar questions myself. improvement in the efficiency of a process. the known sequences of the mouse and However, I am not aware of any other more I first reported these results at a workshop human genomes and, third, the investigator successful means of dissecting complex bio- organized by Frank Ruddle in 1978, held has complete control of how to modulate the logical phenomena into manageable, under- in Estarreja, Portugal. The extremely high chosen genetic locus1. This last advantage standable components. It is to be hoped that efficiency of DNA transfer by microinjec- provides the investigator with the ability to through the summation of numerous such tion made it practical for investigators to use design the genetic modification of the chosen components, the desired level of clarity of this procedure to generate transgenic mice locus so as to best address the specific bio- even very complex biological phenomena that contain random insertion of exogenous logical question that is being pursued. Such will be achieved. Furthermore, when more DNA. This was accomplished by microin- modifications could include the creation of holistic approaches have been applied to the jecting the desired DNA into nuclei of 1-cell null mutations or HYPOMORPHIC MUTATIONS, analysis of the same processes, they have so mouse zygotes and allowing these embryos the introduction of reporter genes to follow far failed even more miserably, in my view, to come to term after surgical transfer to gene expression or determine cell lineage, to provide significant understanding of these foster mothers4–8. Following this work- and/or manipulation to restrict the effects of complex topics. shop, Frank Ruddle rapidly championed the mutation to any desired group of cells or The initial development of gene targeting our results throughout the mouse research organs (spatial restriction) or to any chosen in mice required the solution to two basic community. temporal period during the life history of the problems. The first and foremost was how to The efficient transfer of the HSV-tk gene mouse (temporal restriction). Surprisingly, produce specific mutations in a chosen gene into cells by microinjection required the NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 507 nnrg0605-cappe.inddrg0605-cappe.indd 550707 © 2005 Nature Publishing Group 117/5/057/5/05 22:28:21:28:21 ppmm PERSPECTIVES a these short sequences increased the trans- Incidentally, we arrived at the above conclu- forming capacity of the injected HSV-tk sions before knowing that gene targeting gene by 100 fold or more3. I showed that the would soon be feasible in yeast12. enhancement did not result from independ- In 1980, I submitted a grant proposal to ent replication of the injected HSV-tk DNA the US National Institutes of Health (NIH) as an extra-chromosomal plasmid, but that to test the feasibility of gene targeting in the efficiency-enhancing sequences were mammalian cells, and these experiments either increasing the frequency of exogenous were rejected on the grounds that there was DNA integration into the host genome, or only a remote probability that the newly increasing the probability that the HSV-tk introduced DNA would ever find its match- gene, once integrated into the host genome, ing sequence within a host cell genome. was expressed in the recipient cells3,9. These Despite the rejection, I decided to continue experiments were carried out before the con- this line of experimentation. We knew that cept of gene expression ‘enhancer sequences’ the frequency of gene targeting in mamma- had emerged and contributed to the defini- lian cells was likely to be low, and that the tion of these special DNA sequences10. As insertion of the targeting vector at various I describe below, the emerging concept of random sites in the genome, other than the b enhancers profoundly influenced our con- target locus, would be far more common. tributions to the development of gene tar- We therefore proposed to use selection geting; they alerted us to the importance of to eliminate cells that do not contain the using appropriate enhancers to mediate the desired targeted homologous recombina- expression of newly introduced selectable tion products. The first step of this scheme genes, regardless of the inherent expression required the generation of mammalian cell characteristics of the host site to which they lines with random insertions of a defective were targeted. neomycin resistance (neor) gene containing However, the observation that I found either a deletion or point mutation (FIG. 2). In most fascinating from these early microinjec- the second step, target-vector DNA, carry- tion experiments was that when many copies ing defective neor genes with mutations that of the HSV-tk plasmid were injected into cells, differed from those present at the target site, although integrated randomly into one or two was introduced into the recipient cell lines. chromosomal sites, they were present within Homologous recombination between neor those sites as a highly ordered head-to-tail sequences in the targeting vector and the Figure 1 | Generation of an integrated, concatemer (FIG. 1). We reasoned that such recipient cell chromosome could generate a head-to-tail concatemer of multiple copies of highly ordered concatemers could not be functional neor gene from the two defective a plasmid following its injection into nuclei generated by a random ligation process, but genes, producing cells that are resistant to of cultured mammalian cells. When multiple copies of a DNA sequence (arrows) are introduced could be generated either by replication (for the drug G418, which is lethal to cells that r 13 into mammalian cells (a), they are efficiently example, a rolling-circle-type mechanism) lack a functional neo gene . integrated into one or a very few random site(s) or by homologous recombination between within the host genome as a concatemer (b). the newly introduced plasmid molecules. neor– Sequence analysis of the concatemer shows that We proved that they were generated by the multiple copies of the DNA sequences are not homologous recombination11. This was the randomly oriented within the concatemer, but first demonstration that mammalian cells rather are all oriented in the same direction (a head-to-tail concatemer; b). We proved that could mediate homologous recombination such highly ordered concatemers arose through between newly added exogenous DNA neor– homologous recombination11. This was the first molecules. This conclusion was significant demonstration that generic mammalian somatic because it showed that mammalian somatic cells contain the enzymatic machinery for cells, in this case mouse fibroblasts, con- efficiently mediating homologous recombination. tained an efficient enzymatic machinery for neor+ mediating homologous recombination.