Where do you work now?
Smithies: I work in the University of North Carolina in the Department of Pathology and Laboratory Medicine. I always get these mixed up, and so I’m really a pathologist now, officially, although I’m in actual fact — have been for a long time — and I’m now a molecular geneticist… is the best description of what I am.
Kucherlapati: Oliver, were there any early incidences in your life that sort of attracted you to science?
Smithies: Oh, I think probably it was a comic strip. There was a comic strip that had an inventor in it, and I thought that was neat stuff and I’d be an inventor. And that’s what I’ve been.
Kucherlapati: When was this?
Smithies: Probably seven or eight, something like that. As long ago as that, ever since I can remember, I wanted to be an inventor.
Kucherlapati: So you were always interested in science.
Smithies: Yeah, I think that’s absolutely true.
Kucherlapati: Are you still interested in science?
Smithies: Oh yes, I can’t stop it. I have a little story about science that I think tells the story. A scientist loves the work but shouldn’t be so engrossed in it that there isn’t anything else. So a scientist should have three things in life. Should have his or her science, should have a good hobby and should have a family life. So it comes, now we’re talking about Saturday, it’s the weekend, you wake up, what do you want to do? These three competing things: you want to go and do an experiment, you want to go flying, and you want to take your wife out to lunch. And if you’re lucky you can do all three; I did all three last Sunday, or maybe the Sunday before that I think.
Kucherlapati: Oliver, one of the early things that you did in science is to develop a method for separating proteins by starch gel electrophoresis. Can you tell me a little bit about how the ideas came about developing this method?
Smithies: Yes. I mentioned that I went to Canada for my first job now after my post-doc. And the person I went to work with was named David Scott. He’d been one of the earlier investigators of insulin and the purification of insulin, which was discovered in Toronto, which is where he was and where I went to work. And he said I could work on anything that I liked when I went to work with him, as long as it had something to do with insulin. I decided I would look for a precursor of insulin… in trying to develop a method of detecting what I thought would be very like insulin but would not be insulin because I didn’t think insulin was the natural product.
I was right, but never discovered the product. The real thing… I had a problem with the insulin… it wouldn’t migrate in my electrophoresis medium, the medium I used to separate proteins, which was filter paper soaked with a buffer. You put the protein on this filter paper and pass it in an electric current. It should migrate down the filter paper and separate from other things. But insulin stuck to the paper, terribly stuck to the paper, and it would just unroll like a carpet. So if you had a small amount it would unroll to here, if you had more it would unroll to here. And it was hopeless.
Then I heard of some guys who in the Hospital for Sick Children were using starch grains for a supporting medium for electrophoresis. And to give you an idea of what that’s like, it’s like taking a sand pie and filling it full of sea water so it’s sort of semi-solid, but there really is liquid in it and your protein can migrate all around the different grains and separate. Well they were doing this with starch grains and they had no problems with things sticking to the starch grains. But in order to find the proteins, they had to cut this sand pie, if you like, or this starch block into 50 chunks and measure the protein in each chunk.
That meant 50 protein determinations to do just one electrophoresis. Well, I didn’t even have a dishwasher, I didn’t have a technician, I couldn’t afford anything like that if I was going to do science, but I thought this was neat that it didn’t stick to starch.
Then I remembered a key thing, and this is part of my history that all of this work stems from in a way: I remembered helping my mother to do the laundry when I was about 12, and when she starched my father’s clothes she made the starch by boiling water and whatever. And then after you tied it up at the end it was a jelly, and I thought well, if I just cook the starch into a jelly, then I won’t have to slice it like this and I can stain it. And so I can cut out all this 50 protein determinations… and so it was a lazy man’s approach, and it invented this new method of electrophoresis that proved to be quite powerful.
Kucherlapati: Oliver, you said that you initially trained as a physiologist, and now you’re a geneticist. How did you transition from being a physiologist to a geneticist?
Smithies: Well, I always had this problem with deciding what I wanted to be. So when I went to college I couldn’t decide whether I wanted to be a physicist. I won my scholarship basically on the basis of my physics and math, or whether I wanted to go to med school. And I dropped out of med school and went into physiology, and then did chemistry and combined the two. In a way I think I’m very fortunate because I was never frightened of biology and I was never frightened of chemistry. Often enough people are good at one or the other and frightened of the other one.
So it gave me this feeling of being able to do both, which I’ve always treasured. Think perhaps when I mention my tutor again, Sandy Ogston was his name, I didn’t say his name, but Sandy Ogston always had a very broad view of science and taught that way, and got you to think that way. So whether it was physiology or biochemistry or molecular or DNA or protein didn’t make any difference — you just think the same sort of thoughts, transfer the medium.
Kucherlapati: So Oliver, you continue to do experiments now?
Smithies: Oh yes, every day pretty well.
Kucherlapati: You enjoy doing that?
Smithies: Oh very much. It’s a marvelous thing about science. I think that people, maybe people who aren’t in science don’t understand that science isn’t really achieving some great aim — it’s a lot of little steps. And you have to enjoy the little steps every day so you have to enjoy the experiments you do. It’s not so critical that they work, only that you felt that you did a good job and you’ve got… I know it didn’t work but now I think I know why it didn’t work. So you change the experiment and you do the experiment and if you’re lucky, you get lots of experiments and have the enjoyment. I like to do experiments that I get an enjoyment each day. So most of my experiments are relatively short. They might have a three-year or five year total span, but each little experiment is short.
Kucherlapati: Oliver, you’re really very well known as a toolmaker. Can you explain why people consider you as a toolmaker?
Smithies: Well, I suppose I really think I am a toolmaker; the starch gel electrophoresis, which was the one we were talking about with cooking the starch, turned out to be very powerful. It was the first of the high-resolution gel electrophoresis systems for separated proteins. And molecular biologists, I don’t think any of them would function as well as they can today without polyacrylamide gel electrophoresis, which was the immediate following of that tool. It enables you to solve the problem you have, but it gives other people a tool also.
So you get enjoyment from not only your own use of the tool but other people’s use of it. Probably the most important tool that I helped to develop was the means of altering genes and cells, which is essentially what the Lasker Award is about.
Kucherlapati: Oliver, you’re being honored for the development of a powerful technology for manipulating the mouse genome with exquisite precision. So can you tell us about what this technology is?
Smithies: The easiest way to describe it is to think of DNA as being a set of instructions. The DNA in each cell in your body has a whole set of instructions, and these are written out in an alphabet. It happens that the alphabet of the gene is only four letters and we normally use an alphabet in English anyway of 26 letters. But you can convey this information in a lot of letters. So it’s a very linear set of messages.
In fact, it’s rather a lot of messages. If you think of it in terms of a book, it would take a thousand books, each with a thousand pages to represent every letter that’s present in the message of the human genome. And my thought was that we could match DNA coming from outside a cell with the right place in this book, if you like, or in these thousand books, on the basis of one piece of DNA being able to recognize another.
I knew about that from the work that I’d done with the gel electrophoresis. Quite a bit of that work had been related to a protein called haptoglobin, and there were genetic differences in some people, not the same in others. That turned out to be related in part to DNA finding a partner and what we call recombining, exchanging partners, if you like. So here’s how I think of it in trying to tell people the sort of thing that we’re doing: Supposing we have a book that has in it, somewhere in the book, “I think we should go out to lunch in New York today.” That probably only occurs once in the book, maybe a long book.
If you wanted to find that sentence and change its meaning, you take a piece of DNA and you synthesize it or make it in a certain way that it says, “I think we should not go out to lunch today in New York today.” Now you put a new DNA into the mixture and ask it to find the partner. It goes along and it looks around everywhere, and there’s only one partner it can find. It finds, “I think we should go out to lunch in New York today.” But it’s got a new twist to it because it’s put in a little bit extra, it’s put in “not.” So if those two exchange places now, then this book says I think we should not go out to lunch today in New York. It’s completely changed its meaning only by introducing one piece of DNA that you find the target and then exchanging partners, as it were. That’s the principle of what we have been doing.
Kucherlapati: Why did you think of doing these types of experiments, Oliver?
Smithies: Well, you know, it was a slow process really, in a way, because my early work with the starch gel electrophoresis had been working with proteins. And then later as we learned how to do these things and were taught by other scientists, we learned to start thinking about DNA instead of proteins. So I began to study and my people in my lab began to study the differences between different genes now at the DNA level.
We found out when we examined genes that quite often we could find evidence that one gene had given the path of its sequence to another related gene, so it was as if there were two genes and one said, “I’m not going through, the New York business,” and the other one said the slightly different message, and yet they exchanged information. This happens naturally and we found evidence of this. So it seemed to me that it was a constant, continuing sort of thought that we ought to be able to use this process to help cure genetic… help cure genes, if you like. To take what we would now call gene therapy.
So I thought that if I can introduce DNA into a cell, I might eventually be able to alter a gene that’s in the cell. My initial thought was to try to do this for sickle cell anemia so that the gene I was interested in, therefore, was the same gene that affects… that is different in individuals who have sickle cell anemia, the betaglobin gene, the gene that codes for a part of hemoglobin. I’d always been thinking it would be good to be able to do this. We had plenty of supply of the good gene, as it were, and we knew there were individuals who had a gene that was damaged, and surely we ought to be able to put these together using this message searching that I was talking about, this information searching. But I couldn’t think how to do it, and what would make it work.
Then I was teaching a class in genetics in Wisconsin, and I was teaching about a paper that had been published — I think it was in something like April of 1982 — and in this paper, the first author was Goldfarb and the last author was Michael Wigler. They described a method that they had used to isolate one of the first genes related to making a cell into a cancer type of cell. It was very neat and very complicated, but I suddenly realized I could use this same principle to see if I could make one piece of DNA find a partner. The principle that that came out of that, and I wrote a page in my notebook about it, was fairly simple in idea.
But let’s go back to the “not” example. If I have “not” in the incoming DNA and there is no “not” in the target, then if I can get that “not” into the target and show that “not” is there, I know I’ve hit the gene. I used a piece of DNA, which says — if I can remember my metaphor well enough — I used a piece of DNA that said that “I think we should not go out to” and didn’t go on. But that’s enough to find “I think we should go out to” to change it to “not.” And now if I can find a “not” next to “lunch today in New York,” I know that I hit the target. I have to find that piece of DNA that now has “not” next to “New York.” Because the “not” was on the incoming DNA and the “New York” was on the target, and they were separate.
But if I’ve hit the target, then “not” would now be near “New York,” and that was the principle of the method that I invented. It took three years and it took your help, Raju — you know very well — I’m going to tell whoever is listening that Raju and I have been collaborating in science for I think 30 years. Is it about 30 years, Raju? I think it might be more. Anyway, we’ve been collaborating a long time, and so when I wanted to do this experiment, the first person I turned to for help was Raju. So together we did this experiment.
Kucherlapati: Oliver, do you remember the day when you were able to have convincing evidence that this event took place?
Smithies: Yes, I do very well; it was it was a spectacular day. The page I told you about that I wrote in my notebook after reading the Goldfarb and Wigler paper was some time in ’82, and then we took about three years of work to get to the proof of the correctness of the idea. And the final proof was to have some cells that we thought by indirect means — we could assume by indirect means that we had achieved this targeting. And finally, we got down to these cells and purified the DNA from these… my post-doc Ron Gregg purified the DNA from these cells. He ran a gel to separate the DNA and we knew that if we had achieved our aim we would find a piece of DNA that, if I remember correctly, would be seven kilobases long, seven units long. Whereas if we hadn’t been successful, it would be 11 long.
So this gel had to be run, and then I got the privilege of developing the film that told us what had happened in that experiment. I remember very well in the dark room thinking “now we’ve been working for three years on this with indirect evidence and now we’re going to know the truth.” This is like being a pilot flying in the clouds. I do that, as an instrument-rated pilot, and you fly a certain set of indirect indications, needles and things which let you know where you are, and you fly with the needles in such a way that you keep the needles exactly crossed like this. If you do that, you go down this line and then you come out of the clouds at the end of this line and there’s a moment of truth. Either the runway is there or it’s not there, and so this film, in a way when it was developed, it was like finding your runway.
And there it was, it was right and I went and told people at the scientific meeting and everybody at that meeting whenever they had their final result they would point to it and say, “And there is my runway.” So I remember very well.
Kucherlapati: Oliver, you’re being honored for applying this technology for manipulating the mouse genome. So how did that come about?
Smithies: Well, in a way it came about because we were not successful. We were not successful in achieving these modifications of genes with a high frequency; it was really very rare. So instead of what will be needed for gene therapy, let’s say a ten percent success rate at least or something like that, or maybe one percent, we were only succeeding one in a million times. And so that wasn’t really very useful for gene therapy.
But then I went to a meeting — a Gordon conference — I heard an investigator talk about these new cells that had been isolated by Martin Evans. These cells were called embryonic stem cells. Now many people now know what embryonic stem cells are, but at that time it was still very early days, back in the ’80s. I heard about these cells that Martin Evans had isolated, embryonic stem cells.
I thought well, my gosh, even if it’s very rare, we can do one of our experiments with embryonic stem cells and find the one in a million that has been altered and use that to make a mouse that has now a gene altered in a way that we wanted. And it doesn’t matter that it’s rather rare and rather difficult, because you can in a sense sift through all of the cells and find the one that’s been correctly altered and then return it back to a mouse, as Martin Evans taught us how to do, and get a mouse with the altered gene. So that’s how we came to be doing that, because it was an obviously useful thing to do with this technology which didn’t quite solve the problem I set out to do. I’m still working that, but maybe one day.
Kucherlapati: Oliver, the paper describing the first gene targeting was published in 1985. What has been the impact of this discovery over the last 16 years?
Smithies: Well, I suppose the end fact of it has been rather remarkable to me. But I don’t want people to think that I consider this just my invention as it were, my tool, because Martin Evans was there and Mario Capecchi, who’s being recognized, he made huge advances in making it much easier to do this than my methods have been. But together, as it were, we made a contribution to this. It’s enabled people to do many things that they had not been able to do before. It enables them to alter genes in such a way as to replicate human conditions, human genetic conditions that are harmful or for the individuals maybe even much more than harmful. So we could replicate some human conditions by making these changes in genes.
For example, one of the early ones, which we did, was to make a model of cystic fibrosis. This is a result of one gene being defective. So we made a mouse in which we made this gene defective. Bev Koller, my post-doc at the time, was a valiant worker in this, worked very hard to make that mouse… It’s led people to have a method of understanding what genes do when they don’t know anything about them. Here we’ve isolated a piece of DNA, and we’ve got this thing that looks like a gene there, but we don’t know what it does.
So one of the best experiments to do under those circumstances is to find out what happens when it’s missing, when it’s not working. And this gives one the capability of doing that. So many people use it to find out what genes do, because if you take it away what goes wrong? Well, if you take off the steering wheel of a car, what will happen to it? And you find out pretty soon that the steering wheel is essential to steer the car. The same way if you take out some essential gene, you find out that the animal won’t do or can’t do whatever was appropriate for that particular gene. Many, many several thousand of papers have been published using this technique since.
Kucherlapati: Oliver, all the techniques that are used to manipulate the mouse stem cells — can they also be applied to manipulate human stem cells?
Smithies: In principle the answer is yes, but in practice the answer would be “it would be foolish.” Because as I mentioned, we have to sift through millions to find the right one. Usually we get what we expect, but we don’t always get what we expect. And that means that it’s not really a fully controlled process, and it would be very foolish in my mind to try to use that in embryonic stem cells in humans at this stage. At least embryonic stem cells, unless they were being used for what we might call local effects, something that you could be sure if you weren’t right it wouldn’t cause harm. And I think maybe I’m modifying my statement, you see — if it’s used appropriately it could be valuable, but if it’s used inappropriately, it will be foolish.
Kucherlapati: Without thinking about modifying the human genome line, would this technology have the ability to affect the health of humans by modifying the stem cells?
Smithies: Yes. I think the answer is it might have that affect in the end, if one learns how to get stem cells — not necessarily embryonic stem cells, but stem cells from an individual who has something wrong. Let’s say they have something wrong with muscle, muscular dystrophy perhaps. I don’t want to take that too literally, but let’s say that might be an example. Then if one could isolate the stem cell which would replenish muscle cells, or replace, or help prepare, then repairing that stem cell and returning it to that individual would be rather marvelous because one would have avoided many of the difficulties of this type of thing. But it’s rather a long way away, I think — it doesn’t mean we shouldn’t work hard to get there, but don’t expect it tomorrow.
Kucherlapati: Oliver, you were saying earlier that, those particular technologies, if they’re used appropriately, could have great significance or benefit to human health. So what, what do you think the ethical implications of this gene-targeting work in human stem cells?
Smithies: I think so much of this is tied up with a person’s religious beliefs as to what’s appropriate and what is not appropriate, that I can’t give an answer that I think is other than personal. And maybe somebody else might not think the same way. I think that if we can help people, then it’s appropriate. If we do harm to people, then it’s not appropriate. I don’t think there are any moral implications in doing it; it’s whether we help or we hurt. If we help it’s appropriate; if we hurt, it’s not appropriate.
Kucherlapati: Oliver, thank you for taking the time to talk to me about your experiences and how you’ve gotten into this research, and how excited you are about the work that you do. I think that you would be excellent model for young scientists to follow in the footsteps of people like yourself. You’ve always been excited about science and have the ability to transmit that excitement to everybody.
Smithies: Thank you, Raju.
Key publications of Oliver Smithies
Smithies, O. (1955). Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochem. J. 61, 629–641.
Smithies, O., Connell, G.E., and Dixon, G.H. (1962). Chromosomal rearrangements and the evolution of haptoglobin genes. Nature. 196, 232–236.
Slightom, J.L., Blechl, A.E., and Smithies, O. (1980). Human Fetal Gy- and Ay-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell. 21, 627–638.
Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati, R.S. (1985). Insertion of DNA sequences into the human chromosomal B-globin locus by homologous recombination. Nature. 317, 230–234.
Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton, D.W., Thompson, S., and Smithies, O. (1987). Targeted correction of a mutant hprt gene in mouse embryonic stem cells. Nature. 330, 576–578.
Smithies, O., Kim, H.-S., Takahashi, N., and Edgell, M.H. On the importance of quantitative genetic variations in the etiology of hypertension. Kidney Int. (in press, 2000).