The Birth of Gene Targeting
Transcript of Part 1: The Birth of Gene Targeting
00:00:14.27 What I'd like to discuss is the birth of gene targeting, 00:00:19.16 particularly our contribution to this field. 00:00:22.20 First of all, what is gene targeting? It's a method, essentially, of being able to 00:00:29.01 change any gene in any conceivable manner in an organism. 00:00:33.28 And our particular organism is the mouse. 00:00:36.15 And so what we want to do, the mouse has many genes, 30,000 genes. 00:00:41.15 And this allows one to selectively inactivate a particular gene 00:00:45.07 and for example, if a little finger disappears 00:00:49.06 then we know when the program for making a little finger is. 00:00:51.13 And then that way, be able to deduce, essentially, what each gene 00:00:57.01 is doing by what outcome to the mouse is...when we modify a particular gene. 00:01:03.26 So how is this done? 00:01:05.29 The experiments actually started back in the 1970's. 00:01:12.03 Richard Axel and Wigler had shown, essentially, if you make a 00:01:20.00 precipitate of DNA and put them on top of cells, the cells eat the DNA 00:01:25.08 and then a certain amount of it would then go 00:01:28.25 into the genome and be functional. 00:01:30.16 For example, if a cell is thymidine kinase minus, this is an enzyme that's required for 00:01:36.23 thymidine uptake. So if that gene isn't there, they can supply it exogenously. 00:01:44.09 And they add...make a precipitate, give it to the cells, and about 00:01:47.29 1 in a million cells actually, then, acquires this gene in functional form 00:01:52.27 and becomes thymidine kinase positive. 00:01:56.22 So, first of all, we thought, well, perhaps, if we actually made needles 00:02:01.26 very small needles and they were like microinjection needles 00:02:05.10 like a hypodermic, we could actually direct the hypodermic right into the nucleus of the cell 00:02:11.05 and thereby plant the DNA into the nucleus and maybe that would work much more efficiently. 00:02:17.12 And that turned out to work...instead of the efficiency being 1 in a million 00:02:22.28 it is now 1 in 3. So 1 in 3 cells acquire the cell in functional form. 00:02:28.07 But the DNA went randomly into the genome 00:02:31.29 it didn't go into a very specific place. 00:02:34.17 So we repeated those experiments and what we noted is if 00:02:39.18 we put in multiple copies of the same DNA, 00:02:44.11 what we found is that, again, that...all of that DNA went randomly into the genome 00:02:49.17 but something very unexpected was seen. DNA has a direction; you read 00:02:54.12 it from say left to right. And so, what we found is that all the DNA molecules 00:03:00.22 were lined up next to each other in what we call a concatemer, 00:03:03.28 a head to tail concatemer, they're all in the same direction. 00:03:07.20 Now, randomly, that's impossible, because we would put in a thousand copies 00:03:12.20 and a thousand copies would all be head to tail, head to tail, head to tail. 00:03:16.20 So there were only two possibilities for how this could happen. 00:03:22.01 One is that that the...one would act like a template and then like a sausage machine 00:03:28.10 and then turn out more and more copies and it would all come out as one 00:03:33.09 large concatemer, again head to tail. 00:03:36.00 The other is by a process called homologous recombination. 00:03:40.08 Where in essence, two molecules which have the same sequence can be split 00:03:46.28 and be put together again and then again would have a head to tail concatemer 00:03:50.29 by homologous recombination. 00:03:53.09 And we were able to prove that, indeed, it was homologous recombination. 00:03:57.22 First of all, that told us that the cells had homologous recombination. 00:04:02.01 Second, it told us it was actually fairly efficient. 00:04:05.05 You add a thousand molecules that are all stitched together by 00:04:07.28 homologous recombination. So that was quite remarkable. 00:04:11.25 The other thing that was remarkable is that we were using fibroblasts. 00:04:15.25 These are cells, for example, that are present in our skin. 00:04:19.12 And that was unusual because, people previously knew about 00:04:24.25 homologous recombination, but they thought it had to do with sex. 00:04:28.26 It had to do with parents, you know you always get a chromosome 00:04:34.02 from your mother and a chromosome from your father and then instead of getting 00:04:39.13 a whole chromosome yourself from one from your father and mother 00:04:42.28 then they're split into many, many pieces and stitched together again by 00:04:47.23 homologous recombination so that you get a much more variation essentially. 00:04:52.11 Instead of getting a whole chromosome, 00:04:53.14 you get a chromosome that's made up of parts 00:04:55.21 from both your father and your mother. 00:04:58.08 And then that way the variation of gene copies of gene variation that you get from 00:05:03.12 your two parents is much greater than if you got a whole block of chromosome, 00:05:07.03 one chromosome from your father and so on. 00:05:09.01 So it mixes up and so that makes every sibling are different from another 00:05:13.21 and simply the combination of genes that you're acquiring from your father and your mother. 00:05:17.18 But we were seeing it in skin cells. 00:05:20.26 Fibroblasts which were derived from, for example, skin. 00:05:25.00 And so that wasn't expected. 00:05:27.12 So what that told us is that the machinery is there to do homologous recombination 00:05:33.13 in any cell of the body. 00:05:35.24 So that was the beginning of gene targeting. 00:05:39.25 Now, we wanted to go to the next step. We not only wanted simply to 00:05:44.24 have homologous recombination between exogenous DNA molecules 00:05:49.03 but we wanted to be able to have homologous recombination between a chosen gene 00:05:53.26 that we're introducing from the outside that we've modified some way 00:05:57.28 and then put it in the cell, it would find, essentially, 00:06:01.18 its cognates of the same sequence in the genome 00:06:05.02 exchange information with it, and then any modification that you could 00:06:08.29 create in the test tube would now be present in the chromosomes of the living cell. 00:06:14.26 So that was the intent and we...that was what we wanted to do right away, 00:06:20.11 and also we actually even wanted to do it in mice. 00:06:25.08 Unfortunately, it took about ten years to develop this. 00:06:27.27 So we knew what we wanted to do, we simply didn't know how to get there. 00:06:32.01 And in retrospect, what we're taking advantage of is a machine 00:06:38.17 that normally repairs DNA. For example, sunlight or oxygen radicals 00:06:46.10 that are produced by mitochondria or whatever are destroying the DNA 00:06:50.21 and they make, for example, a double strand break. 00:06:53.27 So what the cell first does is just jam the DNA back together again 00:06:57.26 so that we're not losing thousands of genes 00:06:59.28 that are distal, for example, to the centromere which is required to segregate those genes. 00:07:05.02 However, at the junction where those two pieces of DNA were stuck back together again, 00:07:10.22 a gene is destroyed. However, fortunately we have two copies of this gene, 00:07:15.21 one from your mother and one from your father, if say your mother's copy was destroyed 00:07:21.05 then it can use the information from your father's copy 00:07:26.01 to correct that and that's by homologous recombination. 00:07:29.16 So that's the machinery we're taking advantage of and it's simply 00:07:33.14 present in every cell of the body. 00:07:36.07 So what we had to do is figure out how this machinery worked 00:07:41.13 and then present our DNA to the cell in such a way that it would think it's the right copy 00:07:46.28 and thereby convert, essentially the copy that's in the genome 00:07:50.08 with the exogenous copy that we're adding from the outside that we've modified. 00:07:55.04 And that took about ten years to figure out how to do it. 00:07:58.15 Now the other thing that was not apparent right away 00:08:03.23 was how to then go from cells to mice. 00:08:07.28 And we knew, roughly, how we wanted to do it 00:08:12.12 but unfortunately the cells that we required were 00:08:16.15 embryonic stem cells from the mouse and they didn't exist at that time. 00:08:20.14 And then this is... so now we're roughly in the 1980's 00:08:27.26 In 1984, we already presented data to say, "Well, now we want to do gene targeting... 00:08:36.12 we do gene targeting in cells." We submitted a grant to the NIH. 00:08:40.05 The NIH found that project not possible. 00:08:45.23 They said, "The probability, essentially, of your piece of DNA ever being able to find 00:08:50.28 that same sequence in 3 billion base pairs is impossible. 00:08:56.03 I mean, the frequency would be much to low and therefore it'd never function." 00:09:00.06 And we realized that the frequency was going to be low and so what we were thinking about 00:09:07.18 is simply developing as a part of a selection. An example would be 00:09:12.17 we have a defective gene copy already in the genome, and we'll add a copy of that same 00:09:19.10 gene with a different defect. Either one by itself would not be functional 00:09:24.02 but together, by homologous recombination, they could recombine in such a way 00:09:28.01 that now they would give you a functional copy 00:09:30.08 because there are different mutations on those separate genes. 00:09:34.14 And so that allows...and if that gene is required for the cell to survive, then you have a very 00:09:39.29 strong selection that may work...be able to pick up events, one in a million or so. 00:09:45.06 And so that's the way we were approaching it. 00:09:48.27 But still, they were skeptical, they gave us money actually for other projects 00:09:54.07 and what we did was to utilize that money to continue our effort in gene targeting. 00:10:00.26 And fortunately, four years later, we had information that 00:10:04.23 it actually was working. We sent the grant back to the same granting agency 00:10:10.07 and they sent back a pink slip that said we're glad you didn't follow our advice. 00:10:14.26 So, that gives you an idea that, you know, if you have confidence in a particular idea 00:10:22.11 go for it and see whether you can come through. It's also risky in the sense that 00:10:27.00 if four years later, we hadn't had any results we would have been in deep trouble. 00:10:33.00 and unable to obtain other grants simply because we had utilized 00:10:38.11 those funds for something that didn't work. 00:10:40.28 Fortunately, four years later, we were successful and the project continued. 00:10:46.28 The other aspect is, you know, how do you go from cell culture to making mice? 00:10:52.22 And for this, at the time, the most attractive cells were called EC cells, 00:11:00.20 embryonal carcinoma cells. And those are...it's a tumor essentially, that's made up of many multiple 00:11:07.13 cell types and but, within them are stem cells, stem like cells 00:11:12.22 in the sense that they could contribute to the formation of multiple different tissues. 00:11:16.17 And so I was going from meeting to meeting looking at how the progress was being made 00:11:23.02 with EC cells and it was sort of disappointing in a sense that it was working 00:11:27.27 to contribute to tissues of the body, what we call somatic cells, 00:11:32.14 but it wasn't contributing to the germ line. And for us we wanted to go into the germ line, 00:11:37.19 because then, if we ever made a modification, 00:11:40.11 we could then generate as many mice as we want 00:11:43.29 with that modification simply by breeding. 00:11:46.13 But those cells didn't exist. And then, fortunately, at around 1980...late 1984 00:11:53.15 I heard rumors that Martin Evans in Cambridge, England had actually started developing 00:11:59.12 cells that may work. At that time he called them EK cells 00:12:04.08 and those cells, what he did is to isolate, rather than isolating these cells from a tumor 00:12:10.25 he isolated very similar cells from an embryo. 00:12:14.10 And simply used EC cells as the driving force to say, "What kind of cells I want." 00:12:20.05 but now instead of deriving it from a tumor he was isolating them from the embryo themselves 00:12:26.08 and those cells looked like they may be capable of contributing to the germ line 00:12:31.23 and therefore would be a suitable substrate for us to do gene targeting with. 00:12:36.03 So I called up Martin Evans, this was Christmas of, now, 1985 and 00:12:43.01 asked him if I could go to his lab to learn how to work with these cells, and he was 00:12:47.05 very generous and allowed us to go. My wife and I went there, 00:12:51.14 spent several weeks learning how to work with these cells, 00:12:55.04 how to use those cells, then actually how to make embryos in a sense of introducing them 00:13:01.03 to what we call a blastocyst, a pre-implantation embryo 00:13:04.16 and then these EK cells are now called ES cells, would then contribute to the formation 00:13:11.19 of the embryo proper, once we implant it into the mouse 00:13:16.22 but fortunately now these cells were contributing to the germ line. 00:13:20.15 So they were perfect for what we wanted to do 00:13:23.05 in terms of modifying mice. 00:13:26.04 So that essentially gave you the background for us to be able to then, not only 00:13:30.09 go from directing DNA at a particular target in cell culture 00:13:35.27 but now extending it to formation of mice with specific mutations.