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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.

This Talk
Speaker: Mario Capecchi
Audience:
  • General Public
  • Educators of H. School / Intro Undergrad
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: March 2011
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Talk Overview

If you strongly believe in an idea, sometimes you need to take some risks to pursue it. This is part of Dr. Capecchi’s message as he describes the challenges in developing the technique of gene targeting from tissue culture cells to mice.

Speaker Bio

Mario Capecchi

Mario Capecchi

Dr. Capecchi is Distinguished Professor of Human Genetics and Biology at the University of Utah. He is best known for his work on the development of gene targeting in mouse embryonic stem cells. Capecchi was awarded the 2007 Nobel Prize for Medicine or Physiology, with Oliver Smithies and Martin Evans, for his contributions to this… Continue Reading

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Related Resources

Research Paper Discussed in this Talk

Capecchi MR. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet, Jun;6(6):507-12. Review.

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