Disease Models & Mechanisms 1, 197-201 (2008) doi:10.1242/dmm.001966 A MODEL FOR LIFE

The first transgenic mice: an interview with Mario Capecchi

Mario Capecchi recently won the Nobel Prize for contributions to genetics that have catapulted the mouse to the status of the most valuable of all animal models. He has a personal story that is as rich and interesting as his science. Here, he discusses the journey that led him to gene targeting and his vision for the future.

The ability to remove or mutate genes to My thought was that the cells were using assess their function has forever changed endocytosis, where cell membranes engulf the fields of biology and medicine. A exogenous material, to internalize the pioneer of this technology, Mario Capecchi DNA. Cells usually do this with the intent was shaped by a unique childhood that of shuttling the internalized material to taught him persistence and self-reliance. He lysosomes to degrade it, and then use what- was born in Italy during World War II. ever is useful to them. So, my thinking was When he was only 4 years old, his American that most of the DNA in those early exper- mother was imprisoned in a German con- iments ended up being degraded and that

DMM centration camp and soon after he found very little of it was delivered to the nucleus himself homeless and fending for himself where it could function. I thought that if I on the streets of Italy. After 4 years alone, he made very small hypodermic needles and was reunited with his mother and they stuffed the DNA directly into the nucleus moved to the USA. He quickly developed a itself maybe the efficiency would be much passion for physics and his early work with higher. I did it, and it turned out that effi- James Watson further influenced his scien- ciency was much higher. Now one in every tific mind. His discoveries leading to gene three cells, rather than one in a million cells, targeting are rapidly unlocking the myster- incorporated the gene into its genome in a ies of the mammalian genome. functional form. That was interesting and important, but didn’t answer the question In 2007 you won the Nobel Prize for your of how the DNA was inserted into the role in developing the process of gene genome. targeting, in which homologous recom- Wigler and Axel were using a lot of DNA, bination is used to replace an endoge- and what they did was mix plasmid DNA nous gene with one that has been modi- containing their gene of interest with Disease Models & Mechanisms fied. What inspired you to approach such salmon sperm carrier DNA, which was a monumental challenge? cheap to buy, to get the DNA concentration There was a paper by Wigler and Axel [in high enough to form the precipitate. inserted into the genome in the same ori- Cell, 1977] in which they formed a precip- Because I was injecting individual mole- entation. DNA has an orientation, just like itate of DNA and calcium phosphate on top cules, I could choose to put in 1, 10 or 100 reading a text. If I started by putting all of of cells and found that the cells took up the molecules. When I put the needle into the the separate DNA copies into the nucleus DNA. These cells started out lacking a par- nucleus, the solution that I was injecting and they ended up incorporated into the ticular gene that was necessary for survival had a distinct refractive index so I could genome at a single locus in the same orien- in a chosen medium, but if the cells stably watch the solution spread out in the tation, then this process couldn’t happen incorporated the gene into their genome nucleus. I used that information to measure randomly. then they survived; the efficiency was the volume I was adding to the cells. So, I One possibility is that the cell picks up a roughly one cell in every million. Further, could control how much I was injecting and piece of DNA and uses it as a template, like they showed that the added DNA was ran- then I could also control the concentration a sausage machine, to synthesize more domly incorporated into the host cell of DNA that I was injecting and, conse- copies of the DNA in a process that would genome. quently, how many molecules were being end up producing DNA copies all in the injected. I found that the DNA was ran- same orientation. This would be a synthetic Mario Capecchi is the Distinguished Professor of domly incorporated into the genome, but way of generating the observed head-to-tail Human Genetics and Biology at the University of if I put in 10 copies or 100 copies, or even concatamers. The other possibility was that Utah and an investigator in the Howard Hughes Medical Institute 1000 copies, of the same DNA sequence the DNA copies are put together by ho- (e-mail: [email protected]) into the nucleus, all of the molecules were mologous recombination, a naturally oc-

Disease Models & Mechanisms 197 A MODEL FOR LIFE Mario Capecchi

curring process in which similar DNA se- The reason that somatic cells use ho- 2007 Nobel Prize.) I asked if I could come to quences on chromosomes can exchange in- mologous recombination is that every day his lab and learn how to work with ES cells formation with each other. Since homolo- each of your cells receives about 10,000 right then and there. He was very generous gous recombination always maintains se- insults to its DNA. The insults arise from to let my wife, Laurie, and I visit his lab. quence orientation, the DNA that has been oxygen radicals produced in the cells, or When I arrived he said to me, “Nobody newly inserted by homologous recombina- from sunlight, or from everything else that’s seems to be interested in my cells” and I told tion would also be in the same orientation. happening to your poor cells. Often, a DNA him, “They will be, just wait!” I could readily distinguish between these strand gets broken. When it breaks, you not two potential models and showed that only lose the gene at the breakage point, but After you developed this new technology head-to-tail concatamers were generated by upon cell division you lose all the genes that there were limitless genetic unknowns homologous recombination. are no longer associated with a centromere. available for study. Why did you choose At that time, people knew about the The first thing a cell wants to do is stick to focus on the HOX gene family? process of homologous those two pieces of It was timing. At that time, Walter Gehring recombination but they DNA back together so in Switzerland had just characterized HOX thought it was primarily I am a very gene-centric that, rather than losing a genes in Drosophila. As Ed Lewis had restricted to myosis (i.e. guy. Not because genes do thousand genes, it just shown, these genes are responsible for spec- during the formation of everything, but because loses one gene. Once ifying segmental identity in Drosophila. sex cells, sperm and that is done, the Walter Gehring and others showed that eggs). When sperm and genes are the easiest place damaged DNA at the these genes contained DNA-binding egg come together you to be able to dissect junction is tagged and domains called homeo boxes. Amazingly, have chromosomes complex biological the cell can use the copy they also showed that HOX genes not only derived from your phenomena with great of the gene from the existed in Drosophila, but also in frogs,

DMM mother and father, but other homologous chro- mice and humans. This story was breaking these chromosomes are precision mosome to repair it. For at the same time as we were thinking about not intact chromosomes example, if the maternal which gene to tackle with our technology derived from one maternal or paternal copy is broken, then the paternal copy can for modifying mouse genomes. grandparent. Rather, the chromosomes that be used to repair the damaged gene. This was all happening in the mid-1980s. you received from your mother or father are Homologous recombination is the machin- All of a sudden, these HOX genes were extensive mixtures derived from both of your ery that mediates this repair. Every cell in being characterized at the DNA level and maternal or paternal grandparents, respec- your body has to have very efficient homol- identified in many species. Up until then, tively. The extensive exchanges between the ogous DNA repair machinery or it could people knew that, for example, the citric pairs of chromosomes derived from your not survive. acid cycle was going to be the same in all grandparents are mediated by homologous Since the machinery is there in every cell species from plants to humans, so they recombination. This process generates I asked, ‘how can we fool Mother Nature to thought of metabolism as being conserved, much, much greater variation in the DNA use this machinery for our advantage?’ but they didn’t think that the genetic cir- content that is present in each of your chil- What if I could convince the homologous cuitry responsible for development would dren than if your sperm or egg contained recombination machinery that I’m present- be the same among disparate species. The intact copies of chromosomes obtained from ing the ‘good copy’ of the gene to the cell, conservation of HOX genes among many Disease Models & Mechanisms one grandparent or the other. This shuffling and that the cell needs to replace its own unrelated species was the first insight that insures that each of your progeny gets an ex- copy with my copy? Initially, at least, I allowed people studying Drosophila to rec- tensive and unique mixture of DNA derived wanted to present the cell with a ‘bad’ or de- ognize that their work in Drosophila devel- from each set of grandparents. fective copy of a chosen gene, to knock out opment also had something to do with our As I mentioned before, people knew that gene. This thought process led directly own development, and that was very excit- that homologous recombination occurred to gene targeting. ing. during the formation of sperm and eggs, As soon as I saw all of those pieces of I knew I could carry out gene targeting in but I was working with somatic cells newly added DNA lining up in the genome, mice and I thought, ‘What should I do with derived from skin fibroblasts. Scientists and I could prove that this process was me- it?’ The smart thing, I knew at the time, were not aware that extensive homologous diated by homologous recombination, I would be to work in immunology, because recombination also takes place in somatic knew what I wanted to do and I even knew you can isolate B cells and you can charac- cells. I showed that the homologous re- that I wanted to do it in mice. terize them in vitro and in vivo. At that combination machine was present in Unfortunately, mouse embryonic stem (ES) time, many genes involved in immunology somatic cells and that it was actually very cells didn’t exist at that time and they would were being identified but an assay did not efficient, because even if I put in as many be necessary to create mice with the de- exist to determine what they were doing. as 1000 copies of the same DNA molecule signed modifications in their germline. Also, you can wipe out the immune system into a cell, they were all incorporated into Fortunately, about 4 years after we started without killing the animal. From a prag- a single head-to-tail concatamer, all lined all of these studies, Martin Evans’ group matic point of view, immunology would up one after the other in the same orien- were the first to report the isolation of ES have been a perfect system to attack, but I tation. cells. (Sir Evans was a co-recipient of the didn’t enjoy the study of immunology so I

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wasn’t interested in pursuing that avenue. I But, if it’s your PhD thesis on the line, you available. A recent development is the was much more interested in mammalian are going to look at those slides very care- ability to activate genes with light, which at development. However, if I chose to disrupt fully and really see if there is a difference re- least allows the switches to be turned on a developmental gene that functions very sulting from the targeted mutation. and off very quickly. All of these speed- early, it may kill the mouse and I would not This self-reliance also gives you flexibil- related advancements are spurred on by have much to look at. But, I thought that ity. You control the pace. The greater your computers that allow rapid processing of HOX genes were going to be involved later personal involvement, the greater your large quantities of information. In addition in development because, based on Lewis’s commitment to solve the problem properly. to rapid switches, the capture time for work with Drosophila, they were likely to be signals (i.e. reporter genes) will also have to involved with forming the body plan. Does that philosophy influence the way be increased enormously. Technology has So, I thought that HOX genes were you structure your lab? always been important for the advancement working at the right time to provide an in- Yes, I seek diversity. I have people joining of science, but my guess is that in the next teresting phenotype to analyze. My feeling our laboratory from medicine, molecular 20 years we are going to witness remarkable was that the many HOX genes were going to biology, neurobiology and developmental advancements as our ability to process in- have some functional overlap, called re- biology. I always look to see who is missing. formation increases dramatically. dundancy, allowing us to look at that re- There is a constant flux of people coming I am a very gene-centric guy. Not dundancy by combing separate HOX gene and leaving, so I always bring in people because genes do everything, but because mutations by breeding. At the same time, from different disciplines. genes are the easiest place to be able to they functioned late enough in develop- The other advantage of this internal di- dissect complex biological phenomena with ment for the embryos to have probably pro- versity is that everybody looks at problems great precision. By controlling genes you gressed sufficiently for us to decipher what from their own perspective, so if you have can control function and then see what had happened. Even then, after sampling a group of people from different back- happens. You can’t do this any other way. If

DMM opinions from different investigators one grounds they will look at problems from we could control genes at very high speed, lab would tell me, ‘nothing will happen very different vantage points, and I think then we could discover exactly how and because these genes are so redundant that that enriches the whole lab. when the functions for each gene were re- any effect from losing one HOX gene will be quired, even for complex processes such as covered by the presence of another’, and What areas do you think will be most in- laying down memories. This would allow us another lab would say, ‘it’s going to result in fluenced by gene targeting technology in to approach more and more complex prob- a puddle and you are not going to be able the future? lems, particularly the processing of infor- to decipher it. These genes are so important All biological phenomena is mediated or in- mation in the brain. that you are just going to get a mess’. fluenced by genes. Therefore, gene target- I think the mouse is the best model or- Fortunately, the story was in between. It ing will influence the study of all areas of ganism to address such problems. However, doesn’t give you a puddle and there is some biology. Neurobiology, for example, was our brain is much more complicated than redundancy, but there was always a unique surprisingly one of the last disciplines to the mouse brain. It would be nice to be able aspect to the function of each gene. For a adapt gene targeting. I think this reflects the to perform molecular genetics on some- while, gene targeting, particularly in knock- complexity of the nervous system. Complex thing like ourselves or a monkey, but for out mice, received a bad rap in the sense systems often require the use of conditional ethical and pragmatic reasons we cannot do that people would say, ‘I’ve done this ex- mutagenesis and that wasn’t available in the that. Disease Models & Mechanisms periment and I don’t see any phenotype, my beginning. The problem is that if you knock gene doesn’t do anything.’ My retort would out a gene required for liver development, Do you mean that we need to achieve be to simply say, ‘you have to look in the for example, the mouse is going to die, but greater humanization or primatization of right place because every gene has to have a that same gene may also have a function in the mouse? function’. After a few generations, if a gene the brain that cannot be evaluated owing to In a sense yes, in an organ- or subsystem- doesn’t have a function, it is lost by muta- lethality. You have to have a way of separat- specific way. It sounds crazy but there are tion. Every gene has to have a function if it ing these two functions in the animal and reasons to think it may not be as crazy as it is maintained. that requires conditional mutagenesis. sounds. An example for this is that there are There are still problems to overcome in two kinds of bats, big ones (mega bats) and How did you approach something as dif- gene targeting. With current conditional small ones (micro bats). People initially ferent, at this point in your career, as mutagenesis protocols, we can readily thought, as late as the 1980s, that mega bats mouse phenotyping? Did you consult a perform processes that occur in a day, but were derived from primates and micro bats pathologist? we cannot operate within minutes, seconds were derived from rodents. The basis for No, I’m a great believer that the lab should or milliseconds. Yet, our thought processes that conclusion came from looking at the do everything internally. The reason is that operate by millisecond scales. As we are histology of the brain. Mega bats have a when you do it all and watch the story talking, our thoughts are taking shape at brain that looks histologically like a primate develop, you are much more involved. tremendous speeds so, if we are to thor- brain and micro bats have brains that look Further, a pathologist looks at a lot of slides oughly understand these processes, we like a rodent brain. But, DNA analysis tells and doesn’t have the same investment or need switches that can operate at such us that all bats were derived from rodents, motivation to look at each slide with care. speeds, but this technology is not currently suggesting co-evolution of the mega bat

Disease Models & Mechanisms 199 A MODEL FOR LIFE Mario Capecchi

brain in its resemblance to the primate Prize for his contribution to understand- if you can’t put your ideas into practice then brain. Because this co-evolution occurred ing the structure of DNA] lab. It seems they are useless. Lots of people have great in fewer than 70 million years, the major like a pretty exciting time to be working ideas, but you have to convert the ideas into histological differences between our brain in someone’s lab who was a central figure practice. and the mouse brain could not have in- in genetics. My experience also shows that children volved changes in more than a few genes, I had applied to three places: MIT, Cal Tech are extremely resilient. If they are put into a not even 10 genes. and Harvard. I went to Jim’s lab and asked situation, they will work out a solution. You him where he thought I should go. He may look at your own child who is 4.5 years Do you think we’ll be able to create a looked at me and said, “Here! You would old and wonder, ‘how could my child exist primate-like brain in a mouse? be crazy to go anywhere else”. That’s how I alone out in the world?’ But put into that sit- I think it is not impossible at the subsystem ended up in his lab at Harvard and it was a uation many children can do it and do do it. level, perhaps not today or tomorrow, but terrific choice. I should also point out that there is a selec- eventually, subsystem by subsystem. To me, tion process; you would not be talking to such a scenario is more likely than carrying You have unique ideas and have made un- the failures. The only ones that you are out molecular genetics in a primate, which common contributions to science. going to be able to talk to are the ones that would require an enormous investment in Certainly we are all influenced by our have survived. time and cost. Further, personally, for childhood, and yours is one of the most ethical reasons I would have great difficulty unusual that I know. Do you think that What qualities do you think promote working on a primate. When I look at a growing up alone on the streets from 4 to good and creative science? primate, it looks just like me. The ethical 9 years of age influenced you in ways that I think of the process of science as a series issues are enormous. have affected your career? of concentric circles where the small circles Certain aspects may have, for example self- in the centre are where most people are

DMM At one point in your career, you made a reliance. If you are on the streets you have working. As you move further and further big change from physics to biology. What to be able to rely on yourself and gather out to the edge of the larger circles you ap- led you to make such a career shift? everything that is required for survival. You proach science fiction. What you have to do I went to Antioch College, which had a have to get your own food, find your own is find the circle in which you are comfort- work/study program where you study for a clothes and shelter, and so on. So you are able. I like to work near the edge of the quarter and then work for a quarter. At any dependent on yourself and nobody else. I largest circle and hope that I don’t step over time, half of the student body was working think that is the way that I like to look at our that edge, because then I would be wasting and the other half was studying on campus. lab. If we need new expertise, it is better to my time. I like to go out there and work on The jobs were all over the country and de- develop it internally than to farm it out. things that require not only thinking about termined by your academic interests. If you That is where I see the most direct influ- the problem, but often developing the tech- were a lawyer, you would obtain clerking jobs ence. nology needed to solve the problem, which and if you were a scientist you got lab jobs. Another aspect may be intense concen- is why the problem is way out there. The So, I was going all over working in different tration. On the streets you have to concen- further out you go, the longer a project is labs just at the time when molecular biology trate. Particularly in wartime when all re- likely to take you. If it took less time was being born. I eventually ended up at the sources were short, nobody wanted to give someone would have already done it. Such Massachusetts Institute of Technology up their food. To survive you have to steal problems require long-term commitments Disease Models & Mechanisms (MIT). Physics is an elegant discipline; the food, but people with the resources are to the project and an environment that will only discipline that is more beautiful than quite aware that there are many people out support such long-term commitments. physics is mathematics. But experimental there who may want to have a share of their When a new student comes into my lab- physics, particularly particle physics, was in- resources. They are watching their food and oratory I spend several days just talking volving the use of bigger and bigger ma- you have to outfox them. You do that with with the student to find out what their in- chines, and larger and larger groups of sci- patience and observation. It forces you, at terests are. If they are interested and com- entists. I wanted to study a science where the an early stage, to be observant and patient. mitted to a project then they will do a good individual scientist was a more integral par- Science is a mixture of talents, at one end of job and if not, then no matter how hard they ticipant in the experiments and, at that time, the spectrum you are thinking about new try, they are not going to do a good job. It molecular biology was a real draw. I think things and new ideas – flights of imagina- is important to find out what is of interest one reason why there was such a boom in tion. You think about what does not exist to you and what projects are you willing to molecular biology at that time, was that and try to make it so. At the other end of the commit a lot of your time and energy to. people from separate disciplines: chemists, spectrum, science depends on paying at- Science isn’t easy. It requires enormous biologists and physicists, were turning their tention to details. There is a lot of repetition commitment, and a lot of work, time and expertise to biology. It was naive but there to science; for example, an experiment may thinking. But the rewards are tremendous. was a feeling that we could solve any have 10 different steps and each step needs You are generating new knowledge that can problem, no matter how complex. to be done with precision. To be successful make a difference to the welfare of our in experimental science you need to have planet. You did much of your genetics training in talents at both ends of these extreme I build pictures of what I want to do. James Watson’s [who won the 1962 Nobel processes. It is good to have great ideas but Once I formulate the picture, I find out

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what is missing and needed in order to do In terms of molecular genetic analysis, their creative contributions to science. it, then ask where might those missing almost everything we know has been ac- First, although being a member of the pieces exist. This approach requires me to quired from the analysis of bacteria, yeast, Harvard faculty, he was continuously asked look way beyond what I am doing and see C. elegans, Drosophila, mouse, zebrafish, about his progress: ‘What is new?’ The what other people are working on, even in chick and Xenopus. That is, our represen- desire, or need, to give continuous updates completely different fields such as engi- tation of nature has been restricted to just can have the effect of channeling investi- neering, to find out whether they are doing those eight model organisms out of the gators to work on short-term projects, at something relevant to my work. I have to entire biosphere of thousands upon thou- the expense of long-term, high-risk pro- draw some analogies to get an idea going. sands of species. In the past, we have also jects. Dr Capecchi wanted to work on more There is a mixture of almost fanciful ideas, emphasized what is common among these long-term projects like the development of which are close to the realm of being organisms. There may be just as much to be gene targeting in mice, which took 10 years science fiction, mixed with the practicality learned by finding out what makes these or- to develop. Second, he wanted to work in of the details and seeing where the pieces ganisms all different from each other. This an environment that was diverse, collegial will come from and how I might put them area is infinite and could occupy scientists and fun; a place that took its commitment together to make something new. The most for hundreds of years. It is an area that is to including new, young faculty and their valuable information concerning biological currently peaking our interest. Genomes ideas very seriously. Often, many of the problems comes from nature. I see if nature from many different species are very best-known scientific institutions are pre- has done it before and, if so, how has she quickly becoming available but, to date, dominantly filled with senior, established done it? Using this question, I may gain comparisons between these genomes are faculty. In a newly formed institution, there insight on how to do it in our experimental restricted to in silico comparisons. What can be a unique opportunity to develop an setting. If nature has done it, at least I know would really be fun is to be able to access academic environment that benefits from that whatever we want to do is doable, even this wealth of information experimentally. the youthful vigor of a mixture of junior

DMM if I don’t yet know how to do it myself. If So, we need to develop techniques that and senior scientists with unique perspec- nature hasn’t done it, I may be in trouble would allow much greater experimental tives. He indicates that these qualities make because she has had a long time to get it access to this enormous repertoire of bio- the University of Utah an exciting place to done. logical information. extend his career. An example is the involvement of HOX DMM is excited to be able to present Dr genes in regulating finger length. There Interviewer comment Capecchi’s personal story here. We are grate- are people with big hands and people with At the conclusion of this interview, I asked ful to him for discussing his experience in pi- small hands. How does that happen? We Dr Capecchi why he chose, in his early oneering the technology that introduced gene can alter HOX genes in mice and change career, to leave his position at a coastal Ivy targeting to scientific research. We also ap- the lengths of the phalangal bones by League institute and mecca of scientific preciate his candor in addressing some of the about 10%. When you go out and look in discovery for the more isolated environ- additional special qualities and experiences nature, you find that there are organisms ment of the University of Utah, where he that make him so unique. where these small bones are longer than has been since 1973. He made two points Mario Capecchi was interviewed by their body length. That is hundreds-fold that I frequently hear echoed by scientists Kristin Kain, Associate Reviews Editor for longer. How has nature accomplished at other more isolated institutions that, DMM. this? despite their location, are renowned for Deposited in PMC for immediate release. Disease Models & Mechanisms

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