9UNP0185SD_Oliver_Smithies_WordPress

- Well, thank you very much, Fred. I'll return to my involvement with you later. 
(laughter)
 But first I want to say thank you, Lee. I think your vision of the future and science is much broader than mine has ever been. I tend to be this way. And you tend to be that way. It's nice to see the two contrasts. And I just appreciate it. 

But I want also, as I am going to return to this guy, to return to him and his views. So I thought I would do this by, as I call it, turning pages in my life's notebook. And to some degree it will be real pages, and some degree it won't be. But whenever you turn pages in a book, you know, you don't always read them. 
(laughter)
 Sometimes you look at them. And sometimes you quickly turn them over. So there will be a bit of all of those sorts of pages. But I wanted to start with this page, because in a way this encapsulates the beginning and end, as it were, of what I'm going to talk about, because that's my thesis advisor and my undergraduate advisor, Sandy Ogston. I own an enormous amount to him. 

I think he probably had a lot to do with me getting scholarship to go to the college that he was tutoring, a college in Oxford. Certainly, I have a very close relationship with him, because in World War II, housing was short, and I managed to stay and live in his house in a rented room for a while with a friend. And so I became a family member. And he asked me to write his memoirs when he died. The Royal Society of England requires memoirs. We debated who would live longer, but it turned out that I lived longer than he did. I did write his memoirs. And as a result of that, I've read all his papers. I'll come back to that in due course. So anyway, what happened with Sandy first was this. 

This is my first scientific paper. When I actually began it, I was probably 22, or something like that. And I went to Sandy, and we had a weekly meeting, and I had this marvelous scheme for getting energy for nothing. I could go around the cycle and I could make ATP without using any ATP. And I knew it was nonsense. But I didn't know why it was nonsense. And so Sandy and I worked it out. It turned out to be quite interesting. I won't go into the details, except to say that we did publish this. And I had the joy of something like probably 20 years ago now, somebody came to me and said, "Oh, are you that Smithies on Annual Review of Physiology?" 

And I said, "Yeah." He said, "I thought you were dead." 
(laughter)
 Sandy was really very nice, you see. He made this little superscript, Scholar of -- College, which was for me a great honor, and an acknowledge of him of his student. He was always looking after his students. So I did my thesis with him, and I thought I would tell you about it. That was the title of it. And it was published by a chemical journal. And I made this osmometer, a very accurate machine for measuring osmotic pressure. 

I'll show you what it looked like. It was a glorified thermometer in a way, and I was very proud of it. The data was so precise, I had to leave spaces for line to show the data. I'm sorry to say that nobody ever used the method again. 
(laughter)
 I never used it. And it was never quoted in the literature. But I got my thesis. And what was more important, I learned to do good experiments. And I learned to enjoy doing them. 

Sandy used to worry and he said, "Oliver, are you going to get any results?" "Don't worry, Sandy, they'll come." 
(laughter)
 So we had a good time. But anyway, my Post Doc was in the chemistry department here, in physical chemistry. And I worked hard at a method of assessing protein homogeneity by various methods. It was equally successful. 
(laughter)
 It was referred to four times. But only because of the protein I made, nothing to do with the results. 

But it had one treasure in it that I missed, you see, because this is a sentence from it. "The precision of the test was later found to be improved and the labor of setting up greatly reduced by the use of automatic pipettes, but the results he presented were obtained with simple calibrated glass pipettes." So I never published this. And you see, that's 1953. And there it is, and there is my notebook. There is the automatic pipette. It had a disposable tip. It was very accurate. Good to better than a quarter of a percent. 
(laughter)

 I don't need to say anymore, do I? He patented it. Not mine, of course, he invented it independently, but it was the same machine. So where does it really begin? It begins, students, notice, I'm working on January first! 
(laughter)
 I was looking for a precursor for insulin, and was studying the electrophoresis of insulin. It stuck to the filter paper. At that time, that was the only method you could use small amounts. And I was trying all sorts of things. 

They just didn't work at all. I couldn't get it. It would unroll like a carpet. And then, on Saturday... 
(laughter)
 I went to another laboratory. And they told me a new method that they'd been using, where they'd been using electrophoresis in a bed of starch grains. So imagine a sand pile about this big and this deep. Fill it, not with sand, fill it with starch grain. And you have wet solution, it's damp, and protein migrates around. 

But in order to find where the protein is, I have to cut it up into 40 slices and do a protein determination on every slice. One electrophoresis run took you 40 protein determinations. I didn't even have a dishwasher. 
(laughter)
 I couldn't do that. But then I remembered helping my mother to do the laundry when I was a kid. And I noticed that-- I remember noticing that after she had starched my father's clothes, and as we were tidying up, the starch would set into a gel. I thought well, see, if I just set it up into a gel, I can make a gel out of it instead of a moist grains. And then I'll be able to do it by just staining the protein. And that would save an enormous amount of work. 

I wouldn't have 40 protein determinations, I could just stain it. So I went back to the lab and looked around. I used to work in the evenings, so I knew illegally where all the storerooms were. 
(laughter)
 And I found somebody who had some starch, and I took it and cooked the starch. I tried it and the afternoon, you see, Saturday afternoon, and there it was. And the insulin now migrated in a nice way. There aren't any photos, because I didn't have a camera. I have to draw things when you don't have a camera. Anyway, it was very promising and I was pleased. 

And then a couple months later, just for a rough test, I put in some serum into the system, some blood serum, and tried it. I could find, after a few minutes' run. I could find the five-- and that was good. I set up a new experiment at 12 midnight, and ran it overnight. In the morning, I got this result. I could find 11 components. Well, at that time there were only five proteins known in plasma. I didn't do any more work on insulin. I quit immediately and began to work on proteins, serum proteins. Lee told you what he's going to do with them now. 

(laughter)
 So we had the same idea. 
(laughter)
 Only he's going to be more successful, I think. However, there it was. I stopped working on insulin. And my boss, who was a very kind man said oh yeah, that's all right, Oliver, you can work on serum if you'd like to. Then I was about ready to publish. By then, I had tried samples from a lot of people. And these two from George. 

He was a graduate student friend and he became a professional, and eventually president of the University of Toronto. And Gordon Dixon was a distinguished professor in Calgary. But then they were young guys. And I had to get blood from somebody. 
(laughter)
 And they were my friends. Then just about ready to publish and I ran a sample then, and it was very strange. It was most odd. It had many extra components. All these extra bands. 

So what was different? It was the first time I had run the sample from a woman. I thought I found a new method of telling men from women. 
(laughter)
 And I really did, because the next few days, every time I ran a man, it was the same, and every time I ran a woman sample-- I could only do two a day-- I got the other pattern. So I called one "M" and one "F". Then one day about a week later they were reversed. It was a time of sex change operations in Sweden. 
(laughter)
 We had a hilarious time over it. 

It turned out nothing to do with gender. It was inherited. Norma Ford Walker helped me work out the genetics of it. I mention her particularly, because of when it was. That's 53 years ago. She is a woman, she's a PhD. She's head of the Department of Genetics in a hospital. She was some lady. That was way ahead of time. She was one of the most important people in the development of the genetics in North America. 

She was a good teacher. We had a grand time together. And it became clear that the difference that was there was a difference of, it turned out, the protein was haptoglobin. It binds hemoglobin. Just two possible. You could be both copies of gene one, or both copies of gene two. Or, you could have one of each, and you got some sort of complicated pattern in between. And it was obvious that it was complicated. The only protein then that had been solved in terms of genetics was hemoglobin, where there the difference between hemoglobin A and hemoglobin S, the Sickle Cell hemoglobin, had been worked out by some people in England. And there was one amino acid difference. 

And it was obvious that this was more complicated, and therefore, would be of more interest. Not necessarily more interesting but would give us new information. So that's the sort of general message. If you have something complicated, it's likely to be more interesting than something simple. Because you find something new. So we started to do work on that. That was now with George Connell and Gordon Dixon, the two guys I showed you, had by now come back to Toronto, as young assistant professors. We decided we would try to solve this structure. The first thing was to simplify the pattern. That wasn't very common in those days, but eventually, with a series of what I call Saturday morning experiments it was solved. 

Saturday morning are experiments that you can do without any rational. 
(laughter)
 You can do what you like. You don't have to measure anything. You've got to keep good notes, but you can try anything. By the time that worked out, I found that if I use urea and -- ethanol, which Chris Anderson had used for working out the structure of, the forming of RNA, that we could simplify the pattern. But how many of the students have used beta-- in your gels? Stick your hands up. Well, you know what it smells of, don't you? 
(laughter)

 I dropped a bottle. 
(laughter)
 Over my shoes. I put them on the window sill for a week. I only had two pairs. I put them back on again, and went on with my normal life. One of the things I had to do was go to the police station on a completely different matter. 
(laughter)
 I heard these two old ladies talking to each other, "Do you smell it?" "Yes." 

"Do you think it's a body?" 
(laughter)
 I put them back on the window sill. I did wear them again. But anyway, what it turned out was the pattern was more complicated. First of all, there were three genes possible instead of two. There was what we now call 1-F, because it was faster, and 1-S. You could have two copies of gene 1-F, two copies of gene 1-S, or you could have two copies of the gene 2. But you can see from that, that it's funny because the gene 2 seems to be making more product than the gene 1. There were other things that were very peculiar. 

We spent a lot of time trying to solve this. Then one day I said, well, let's see what happens if we believe our results. Because we couldn't believe our results, because one of the results was if you took a mixture of the one gene product, 1-F product and the 1-S product and ran a peptides on them-- We couldn't do protein sequencing-- You ran peptides on them, you got the same pattern as the gene 2 plus something else. The gene 2 seemed to have gene 1-F and gene 1-S and something extra, as well. Then the answer became clear in one of the moments you realize the explanation. It was that there was some sort of event that had put the two genes together. So if I use the letters of the alphabet to represent the 1-F gene, and that's F, and I change the F to S to give the 1-S gene, if I got this sort of event of a recombination, then I could get a gene that was twice as long, made more product, therefore. And it would have both the F peptide and the S peptide. It would have something that was new, because GC doesn't occur in that, and that explained the result. So we were very pleased about this and decided we'd go and talk about it at a genetics conference in Rome. 

By now, I was back in the University of Wisconsin. George and Gordon were still at the University of Toronto. We decided to meet in Rome in the hotel room the night before and talk about the experiment that George was going to do. George was going to do ultra sensitive study of the three proteins with the expectation that the large protein would therefore show in the outer centrifuge. We met the evening before. George had a very long face. Oh, I forgot to mention one thing, but I'll come back to it later. Yes, I'll leave it. The molecular weight determination will provide a direct test of the hypothesis, but George said they didn't differ. The sedimentation constants of the two proteins didn't differ. 

What are we going to do? I said, well, we're going to get up there-- We divided the talk into three parts. George would talk about the purification. Gordon would talk about the peptides. And I would get up and I'd talk about the hypothesis, and that we've done an experiment and it didn't confirm our hypothesis, and we thought the experiment was wrong. 
(laughter)
 And I'll go back and invent another method of measuring molecular weight, so I did. You see, that's September 6. This is when I got back home, October 8. I decided I could measure molecular size by changing the concentration of the starch, you see. 

It's a molecular sieve. If I increase the concentration the starch, the two molecules of different sizes, the large one would slow down more than the small one. So I predicted that if I changed them, the relative mobility of the two small peptides, the two small proteins would be the same, but the larger one would get slower faster. That's the result the next day. I share that partly to tell you students that sometimes you have to believe your results, and sometimes you don't have to believe your results. 
(laughter)
 And that's a tricky business. But consider both possibilities, that your results might be right and your hypothesis might be wrong, or your hypothesis might be right and your results might be wrong. That's important to remember, because as someone said, and you might have been at that meeting, Lee, he once said it's important in talking about hypotheses to know what experimental results to disregard. Sometimes you have to throw stuff out. 

It doesn't fit, oh, 95% fits, I'm not going to worry about that 5% yet. So anyway, that was the way it was. Then I came back to the genetics department. And Jim is here somewhere, stick your hand up, Jim. I have to see you. He is there. Well, Jim was a marvelous geneticist. And he said, you know, what you're talking about is a bit like what's been known for a long time in --. What had been known was that these four individuals, Tice in 1914, Zeleny in 1919, Sturtevant in 1925, and Bridges in 1936, had worked out what was happening to a mutation that occurred that affected the shape of the eye. I'm just going to go back a couple of slides, because I forgot to point out one thing here. 

That is, that this event here, you know, the recombination, is non-homologous. What do I mean by non-homologous? I mean it's taken place between two sequences that are not related to each other. It's not a predictable event. And that's part of what Jim pointed out, that this first experiment, this mutation had occurred in a wild type, changing to this bar shape. It only occurred once. That mutation occurred once. And Tice observed that. Zeleny observed that occasionally, if you were making females, that you would get a return to normal, or something that got more extreme. And Sturtevant showed that that was always accompanied by recombination. 

Bridges provided the final key, and that is that the normal band pattern like this, in the solid chromosome and the bar -- are duplication. You see these occurred twice. But when that one happened, you could predictably, although not very frequently, go back and add it to the single and go to triple. Therefore, that's a predictable event. It suggested the same thing ought to be able to happen in haptoglobin, that we would take these two genes, the two double length genes and we would get recombination. But now it would be different sort of recombination, because it would be homologous recombination. In other words, this crusting over is between sequences that are identical, so it's predictable and it's accurate. We predicted that homologous recombination would lead to a triplication. And in fact, we could find it in every population where the gene 2 was there. You would always find a few individuals who had the triplication, as well. 

It was not possible that that was the same gene transferred from Asia to Europe, or whatever. It was re-arising. So we understood very clearly the difference between non-homologous recombination and homologous. I got fascinated with this homologous recombination. I began to think it would explain everything. Lee might remember this, but back in 1967, we were talking about this. He was talking about antibody variability. And I had the thought that it might be due to crossing over between what I call the gene and the half. Here was the gene, and if this was a region of sequence similarity, not complete identity, so there were differences. You could get crossing overs of various sort, and therefore you could make this gene, the product of the gene vary by recombination. 

I thought it would be a good idea to try it with known sequences, so I put together all the sequences that were known in that way, and tried to work out that it was possible. I even had Elsie Lipinski, a lady friend of mine, made a model of it in a glass bowl that I still have, you see. You can see all these same sequences. We couldn't replicate it in glass. Could we replicate it in real life? I didn't show you, but reference 25 in that paper that had that sequence said, "I am particularly grateful to Dr. Lee hood," etc., for allowing me to use extensive amounts of sequence data from manuscripts in preparation. This young guy, he was a graduate student. Let me use his data to think with. 

That's generosity. That's how science should work. And it is how Lee worked with science. And he published, of course. This is one of his earlier papers on this topic. I think this is when you were a graduate student, isn't it, Lee? All of those sequences marked there, you don't need to know their names, but those are these sequences here. So that's from Lee to me. I'm grateful, Lee. He went on, and began to think about the systems analysis, and he told us a little bit about its rocky pathway in getting people to think it was possible. 

And that's the sort of result he published in his first paper. I can't understand it. 
(laughter)
 I don't know whether he can anymore, he might be able to. He at least showed that it was complicated now, and he knew how to deal with it. 
(laughter)
 So complicated, interesting. But simplicity is interesting, too, sometimes. This is the branched network that Fred was talking about. I thought it might be possible that-- You see, we didn't know the answer. 

You could speculate. And there might be a sort of branched network of DNA like this, and you could go a pathway through there, and you could get the different light change, and you could get the different heavy change, and you could have all sorts of combinations. Well, I did make a glass model of it. 
(light laughter)
 I tried to make a real model, too, using the plasma-- That is what Fred was talking about. I went to talk to him about that. DNA chemistry, as it were, or DNA technology was not up to it, at least in my hands, or in Fred's of that time. It wasn't possible, really. Bill Dove helped. He taught me quite a bit about bacteria phages. 

I always thought that maybe Lambda DV was the name of that, DV means defective varon. But I also think Lambda DV, I think of Bill Dove when I think see DV. It doesn't really have anything to do with that, but he helped me try to do that. So then I began to think, as Fred told you, about moving from proteins to DNA. This was one of our first efforts. And you see Fred's name there. It was really a hybridization of labeled RNA to DNA in agarose gels. This was the ethidium bromide stain of E. coli. This is what you would now call a southern blot, except it was a -- blot that worked. 

But that sentence was added in this paper. A similar hybridization technique has recently been developed by Ed Southern, and his method differs from ours. It worked easier. So Ed, on his southern blots, are well known. And Fred and our RNA blots are in obscurity. 
(laughter)
 We had help, too, then in starting to do cloning of DNA because Fred had developed these safe systems. That was our next job. And this, I was a slave in this work, just to try to learn how to handle bacteria phage and DNA. And Fred was the brain. 

But we were also the subjects, because over here it says that the test was made and nine times ten to the eighth of these bacteria were fed in 250 milliliters, as a milk, to three humans 
(laughter)
 to measure their excretion. 
(laughter)
 And two of the humans are still alive. 
(laughter)
 But we did manage to clone genes and then Phil Tucker, where are you, Phil? Back there. Phil Tucker helped us, because he devised a machine we call the Tucker Box. And it was from his x-ray crystallography experience, where you had a half-silvered mirror. 

You put your bacterial plate down here with all the bacteria phages on it. Because we were big scale, our bacterial plates were cafeteria trays, this big. And they'd take a whole x-ray film for one plate. Then you put the x-ray film on the top and the plate on the bottom, and the half-silvered mirror would let you see which plate was radioactive, and so on. Phil will remember that part of life. But we did manage to clone, then, the beta globin genes, particularly the two fetal genes. That's when Fred and I amicably decided he would work on making the globulins, and I would work on globins. We did notice, again, homologous recombination, because these two genes showed evidence of having exchanged sequences. So I got more and more thinking that homologous recombination ought to be possible to be used practically. Here's a paper of Sickle Cell Anemia, a description of Sickle Cell Anemia. 

I thought, well, this is a mutation of a single base, and we could represent it diaphragmatically this way. The B changes to X, and this is now a sickle cell gene, and that's the mutation. We ought to be able to correct it. We ought to be able to bring in the normal gene, and have crossing over like this, and get back the corrected gene. So that was the thought behind trying to do gene targeting. I wanted to correct a gene. But I didn't know how to do it, and I didn't have any decent idea of an assay. Then, Bob and I taught a course in molecular genetics. There might even be some people here who attended it. I see one person nodding. 

We always taught directly from the literature. This was a paper that came out April 1, 1982. I used this paper and talked about it right away. And what had been done, Mitchell Goldfarth and Michael Wigler had used a method called gene rescue the find a transformer gene. I won't go into the details except to say it was an elegant but very difficult technique, involving the use of suppressive genes and mutant bacteriophages, and repeated cloning, and one thing and another, but it triggered in me the thought that it would be possible. Because, notice April 1, and my April 22, notebook page. Less than three weeks after the paper came out, I put together how to do this. How to use it for gene targeting. And the aim was, an assay to place the corrected DNA in the right place. I needed an assay. 

This is my idea of assay. In fact, the whole scheme is written down on that one page. It is a page, I think, of all the pages in all my notebooks, it's the one I would look at and think that that's a beautiful page. What was the idea? It was a fairly straight forward idea. The idea was if you have something on the incoming DNA, that is not on the target. And something on the target that is not on the incoming DNA, then if these two things come together, you've proved that gene targeting is possible because that's brought together something that wasn't there and put it next to the target that you want to. That must be due to homologous recombination. The whole assay depended upon looking for this recombinant fragment. It required this suppressant gene, and I understood suppressant genes, because of working with Fred. 

He'd used all the mutations, in making his safe viruses. Bill Dove had taught me how to make phage assays. So, I was reasonably confident it could be done. I had to make a target -- I wanted to make a long one, because there the G gamma and A gamma, and other genes that I mentioned before, and the beta gene is a long way away. There were no DNA sequences available then, and there weren't any DNA synthesizers. It was difficult. I won't go through the details, but I'll turn the page. It's a pretty page, isn't it? I don't know what it's doing. But those were on the way. 

And actually, then, we began to do the real thing. Then by now we're a year later, and I got it from-- We did this experiment. Then I noticed that that's June 23rd. That's my birthday. My 58th birthday. It didn't work! I had a good birthday anyway. It was an experiment that didn't work, but it was fun to do. So we kept on. Then began to realize that there was a way to improve things, and that was to use a different construct. 

Because if we used a simple construct and cut it, we could increase the frequency. That was known from yeast work. So we made a simpler construct, all the same elements in it, still trying to do the same thing. That's the construct. And still trying to look for the same recombinant fragment, because the incoming DNA has the suppressor gene on it, but doesn't have the complete beta globin gene. The recombinant fragment will have the complete beta globin gene and the suppressive gene. We began to think about using that. My graduate student Karen Lanz raised a big problem. She said look, you're trying to put this selection in-- We had to have a selectable gene-- into your chromosome. And this is a beta globin gene, again and you're using a bladder carcinoma cell. 

Well, the bladder isn't making hemoglobin. The chances are that that part of the DNA is completely shut away, or something, and tangled away. It's not being used. You might never get the right answer. So you better start doing the experiment in cells that make hemoglobin. And so we began to think, was that possible? And it was because there was a beta globin producing cell that had been made, but they were different. They were in solution. In suspension, rather. They weren't a nice, flat cell sitting on the dish. 

They were all floating around. You couldn't do DNA transformation in those cells in the same way. We used to use a calcium phosphate precipitated DNA. But Hunting Potter came out with a method, which we all use nowadays, called electroporation. But it took a high volt pulse to do this, and there wasn't any apparatus available. So what do you do? Make it. This is my attempt at making that apparatus. This really is the one that was used. That's the test tube right there that forms as a breadboard. 

That's a baby's bathtub. There are some Radio Shack parts. And it actually worked. All of the initial targeting experiments were done with that apparatus. I show it there to get the students, not to make apparatus to save money. You will waste time and waste money if you try to do that. But making apparatus if you can't buy it, then you're saving time. Time, it was two years after that before you could buy an electroporation, so two years was saved by making it. It probably cost as much as buying it, but that didn't matter. It was the time saved. 

We did the experiment, and at the same time, also did some business about getting rid of another worry that we had, because we weren't sure whether we had an experiment that worked. And we got one bacteria phage But we were concerned about had the recombination occurred in the assay, or had the recombination occurred in the cells. We weren't sure which it was. But it was possible to determine which is which by prefractionating the DNA, because the target DNA, I'm sorry, the recombinant fragment would be on an 8 kilobase fragment, whereas the target itself would be an 11 kilobase fragment. If I took my cell DNA after treatment and separated it into an 11 kilobase fraction, and an 8 kilobase fraction, if the positive came from the 11 kilobase fragment I knew that the assay had generated it. And that would be a bad result. If it came from the 8 kilobase fragment, I knew the fragment was already there before it had seen the bacteria phage and the bacteria. In fact, that's what happened. We did that experiment, and Ron Greg did it. I won't go through the details with you. 

We got two positive phages, which you notice that two has been altered, because first there was one, but looking at the film I think another was spotted. In the top corner of one film there was another plaque there that we nearly missed. The big x-ray films, you know, you could easily miss. But it was now clear it was getting right. We could now start to do this by getting pools of colonies. That experiment was done with 1,000 colonies. We could make smaller and smaller colony numbers until this point we had a mixture of about, oh, I think this was about 300 colonies, and pool one. There were eight positives now, so it was quite clear that now we were getting on the right track We could start to do a direct test instead of testing pools of transformed cells, we could take individual transformed cells, grow them up, make DNA and do a direct test, instead of using the assay. That could be done by doing an ordinary southern blot, because as I pointed out, that if we were right, a beta globin probe would light up an 8 kilobase band, and untargeted would be 11 kilobases. There was the result. 

It was one of the colonies now had the 8 kilobase fragments. That was three years and one month after that other page that we got a positive. What have we established? We'd shown that it was possible. Its frequency was terrible. Very low, one in a million cells treated. The assay was unbearable. Never used again. It wasn't any use to do anything to do with curative things. So you had to simplify. 

And the next thing to do was to think about Vassil's work. Where are you, Vassil? There! Vassil worked on the HPRT gene, hypoxanthine phosphoribosyltransferase. It's a very nice gene, because you can assay in a tissue culture for either correcting the gene or knocking it out. If the cells grow on one type of medium, the gene is good. If they grow a different medium, the gene is no good. You could try one or the other. Interestingly enough, Mario also decided to use that gene, too, but it was obvious it was much easier to do a growth test than it was to do that complicated phage assay. We spent two years getting that to work. 

And it worked. But it wasn't any use to be thinking about doing this for gene curative purposes, so what else could we use it for? That's when both Mario and myself contacted Martin Evans, because we knew about the work that he had been doing on using ES cells, embryonic stem cells. Most of you know this story. But these are embryonic stem cell colonies, they're not individual cells. Obtained in this case from a mouse with a cream colored fur. They could be isolated and injected into the blastocyst from a mouse with black colored fur, and then you would get these mice which were striped, with having cells that part of their body came from here, and part of their body came from here. They were mixed genetic type. And they would have offspring, because not only was the skin and the fur patchy, but the testes was patchy. So, there would be parts of the testes which came from here, an parts that came from here. 

That really changed things, because it meant, what are these? These aren't embryonic stem cells, they're mice in tissue culture. You've got mice in tissue culture. You can alter these mice, and do all sorts of things. So we set out to do this. I have this note about thinking about it, and the original cells were received in November, 1985. I look at this note. Martin Evans brought them personally, in his pocket. You see, so never hesitate to ask for help, because other scientists will help you. They're nearly always generous, Lee Hood for example. 

This marvelous community of scientists, at least those whom you want to know. 
(laughter)
 There are occasionally some that you just would rather not know. But that's true of all society. Anyway, we planned to use that, and then what happened, we had help from Tom Doerschman who had already done work with embryonic stem cells. And Nobuyo, my wife, had the right DNA, and that experiment was done. It didn't take three years. It worked the first time. That experiment worked the first time. Sometimes they work the first time. 

And that was the colony that had been corrected, and that was what was left of a colony that had not been. So now we have the ability to alter genes in the mouse. That can lead to all sorts of things. But we can't always depend upon a selectable marker. But by then, such as the HPRT gene, and PCR came out, and it was clear that one could use the same principal, using PCR where you would have one marker on the incoming DNA, and one marker on the target. And then you would get a recombinant fragment that you could find by PCR, which would have both on it, if you used the primer from here and the primer from here. Same problem. No apparatus. Make one. So that was the apparatus, three big water baths. 

I might tell you a little secret. I still use it. 
(laughter)
 It beats all the other machines. But anyway, you can come to my lab and see an old machine like that. So we went on, and Beth Koller used it to make an animal model of cystic fibrosis, knocking out the gene that had been worked on. There was the intestine of this animal. Nobuyo used it to make mice with atherosclerosis. This is the section of the aorta from one of the animals. Look at all the cholesterol clefts. 

A terrible mess. This animal is the best selling mutant in laboratories. It's used all over the world for studying atherosclerosis. Everybody said, before she did this experiment, which I'm not an author on. It wasn't my idea. She said that nobody thought it was possible. Mice only live two or three years. It takes 40 years to get atherosclerosis in a human. Besides which, they have all good cholesterol. They have no bad cholesterol. 

HDL versus LDL. But in fact it turned out that if you hit the right gene they get atherosclerosis on a diet without any cholesterol in it. One of these is sick. And I ask you to tell me which. The answer is you won't be able to tell. Because you can't tell who has atherosclerosis from outside. Her mice are very helpful for this. I did some work on the blood pressure measurements, so that went on. And so I did computer modeling to solve some of the problems. I'm going to end with another visit to Sweden. 

I heard this man talk at an earlier time in Sweden, and he talked about how the kidney works. I thought it wasn't right, what he was talking about. And I'm not going to go into details, except that this is from Ogston again. One of Ogston's papers wrote this equation. It's quite a simple equation. It says the available space, the fraction of available space in a gel is determined by this, where that R is the radius of a protein, little r is the radius of the fibers that make up the gel, and n is the number of fibers per square centimeter. This will predict the fraction of space available. I applied this to the kidney and thought it was neat. And I tried to publish it. I sent it as a little note, but it was rejected by Nature. 

They didn't like the idea. They wrote back saying this part of our journal is only concerned with results, not with ideas. 
(laughter)
 I'm glad they turned it down, because I went on then and wrote a decent paper. And I'm still working on that. That's what I'm working on now. That's the paper. Oh, this is this year, May, and Saturday again. 
(laughter)
 Still working Saturdays. 

But there's a little nice note. The winds were 320 degrees at 20 knots, gusting to 45. And later on in the day. they were gusting to 60. I was going to fly that day. but a 60 mile-an-hour cross wind is a bit hard for a motor glider and I didn't go. I untied it and tied it back up again. But that's a picture out of my notebook. I'm still making experiments. That's in relation to the hypothesis. 

So what's on the next page? I don't know. That's what makes science exciting, isn't it? I'm going to end up by a couple of other things that are needed for making science work. One is you have to have a hobby. I mentioned my flying. The other is, it's good to have a companion, a wife, or however. I met my wife. And she and I have a good time together. But I'm going to end with Sandy Ogston again. 

This is what he wrote. I read this in writing his memoirs, "Science is more than the search for truth, more than a challenging game, more than a profession. It's a life that a diversity of people lead together, in the closest proximity. a school for social living We are members one of another." And I think that that's a marvelous way to think about science. That's where I'll end today. Thank you. 
(applause)