What I'd like to discuss is the birth of gene targeting,
particularly our contribution to this field.
First of all, what is gene targeting? It's a method, essentially, of being able to
change any gene in any conceivable manner in an organism.
And our particular organism is the mouse.
And so what we want to do, the mouse has many genes, 30,000 genes.
And this allows one to selectively inactivate a particular gene
and for example, if a little finger disappears
then we know when the program for making a little finger is.
And then that way, be able to deduce, essentially, what each gene
is doing by what outcome to the mouse is...when we modify a particular gene.
So how is this done?
The experiments actually started back in the 1970's.
Richard Axel and Wigler had shown, essentially, if you make a
precipitate of DNA and put them on top of cells, the cells eat the DNA
and then a certain amount of it would then go
into the genome and be functional.
For example, if a cell is thymidine kinase minus, this is an enzyme that's required for
thymidine uptake. So if that gene isn't there, they can supply it exogenously.
And they add...make a precipitate, give it to the cells, and about
1 in a million cells actually, then, acquires this gene in functional form
and becomes thymidine kinase positive.
So, first of all, we thought, well, perhaps, if we actually made needles
very small needles and they were like microinjection needles
like a hypodermic, we could actually direct the hypodermic right into the nucleus of the cell
and thereby plant the DNA into the nucleus and maybe that would work much more efficiently.
And that turned out to work...instead of the efficiency being 1 in a million
it is now 1 in 3. So 1 in 3 cells acquire the cell in functional form.
But the DNA went randomly into the genome
it didn't go into a very specific place.
So we repeated those experiments and what we noted is if
we put in multiple copies of the same DNA,
what we found is that, again, that...all of that DNA went randomly into the genome
but something very unexpected was seen. DNA has a direction; you read
it from say left to right. And so, what we found is that all the DNA molecules
were lined up next to each other in what we call a concatemer,
a head to tail concatemer, they're all in the same direction.
Now, randomly, that's impossible, because we would put in a thousand copies
and a thousand copies would all be head to tail, head to tail, head to tail.
So there were only two possibilities for how this could happen.
One is that that the...one would act like a template and then like a sausage machine
and then turn out more and more copies and it would all come out as one
large concatemer, again head to tail.
The other is by a process called homologous recombination.
Where in essence, two molecules which have the same sequence can be split
and be put together again and then again would have a head to tail concatemer
by homologous recombination.
And we were able to prove that, indeed, it was homologous recombination.
First of all, that told us that the cells had homologous recombination.
Second, it told us it was actually fairly efficient.
You add a thousand molecules that are all stitched together by
homologous recombination. So that was quite remarkable.
The other thing that was remarkable is that we were using fibroblasts.
These are cells, for example, that are present in our skin.
And that was unusual because, people previously knew about
homologous recombination, but they thought it had to do with sex.
It had to do with parents, you know you always get a chromosome
from your mother and a chromosome from your father and then instead of getting
a whole chromosome yourself from one from your father and mother
then they're split into many, many pieces and stitched together again by
homologous recombination so that you get a much more variation essentially.
Instead of getting a whole chromosome,
you get a chromosome that's made up of parts
from both your father and your mother.
And then that way the variation of gene copies of gene variation that you get from
your two parents is much greater than if you got a whole block of chromosome,
one chromosome from your father and so on.
So it mixes up and so that makes every sibling are different from another
and simply the combination of genes that you're acquiring from your father and your mother.
But we were seeing it in skin cells.
Fibroblasts which were derived from, for example, skin.
And so that wasn't expected.
So what that told us is that the machinery is there to do homologous recombination
in any cell of the body.
So that was the beginning of gene targeting.
Now, we wanted to go to the next step. We not only wanted simply to
have homologous recombination between exogenous DNA molecules
but we wanted to be able to have homologous recombination between a chosen gene
that we're introducing from the outside that we've modified some way
and then put it in the cell, it would find, essentially,
its cognates of the same sequence in the genome
exchange information with it, and then any modification that you could
create in the test tube would now be present in the chromosomes of the living cell.
So that was the intent and we...that was what we wanted to do right away,
and also we actually even wanted to do it in mice.
Unfortunately, it took about ten years to develop this.
So we knew what we wanted to do, we simply didn't know how to get there.
And in retrospect, what we're taking advantage of is a machine
that normally repairs DNA. For example, sunlight or oxygen radicals
that are produced by mitochondria or whatever are destroying the DNA
and they make, for example, a double strand break.
So what the cell first does is just jam the DNA back together again
so that we're not losing thousands of genes
that are distal, for example, to the centromere which is required to segregate those genes.
However, at the junction where those two pieces of DNA were stuck back together again,
a gene is destroyed. However, fortunately we have two copies of this gene,
one from your mother and one from your father, if say your mother's copy was destroyed
then it can use the information from your father's copy
to correct that and that's by homologous recombination.
So that's the machinery we're taking advantage of and it's simply
present in every cell of the body.
So what we had to do is figure out how this machinery worked
and then present our DNA to the cell in such a way that it would think it's the right copy
and thereby convert, essentially the copy that's in the genome
with the exogenous copy that we're adding from the outside that we've modified.
And that took about ten years to figure out how to do it.
Now the other thing that was not apparent right away
was how to then go from cells to mice.
And we knew, roughly, how we wanted to do it
but unfortunately the cells that we required were
embryonic stem cells from the mouse and they didn't exist at that time.
And then this is... so now we're roughly in the 1980's
In 1984, we already presented data to say, "Well, now we want to do gene targeting...
we do gene targeting in cells." We submitted a grant to the NIH.
The NIH found that project not possible.
They said, "The probability, essentially, of your piece of DNA ever being able to find
that same sequence in 3 billion base pairs is impossible.
I mean, the frequency would be much to low and therefore it'd never function."
And we realized that the frequency was going to be low and so what we were thinking about
is simply developing as a part of a selection. An example would be
we have a defective gene copy already in the genome, and we'll add a copy of that same
gene with a different defect. Either one by itself would not be functional
but together, by homologous recombination, they could recombine in such a way
that now they would give you a functional copy
because there are different mutations on those separate genes.
And so that allows...and if that gene is required for the cell to survive, then you have a very
strong selection that may work...be able to pick up events, one in a million or so.
And so that's the way we were approaching it.
But still, they were skeptical, they gave us money actually for other projects
and what we did was to utilize that money to continue our effort in gene targeting.
And fortunately, four years later, we had information that
it actually was working. We sent the grant back to the same granting agency
and they sent back a pink slip that said we're glad you didn't follow our advice.
So, that gives you an idea that, you know, if you have confidence in a particular idea
go for it and see whether you can come through. It's also risky in the sense that
if four years later, we hadn't had any results we would have been in deep trouble.
and unable to obtain other grants simply because we had utilized
those funds for something that didn't work.
Fortunately, four years later, we were successful and the project continued.
The other aspect is, you know, how do you go from cell culture to making mice?
And for this, at the time, the most attractive cells were called EC cells,
embryonal carcinoma cells. And those are...it's a tumor essentially, that's made up of many multiple
cell types and but, within them are stem cells, stem like cells
in the sense that they could contribute to the formation of multiple different tissues.
And so I was going from meeting to meeting looking at how the progress was being made
with EC cells and it was sort of disappointing in a sense that it was working
to contribute to tissues of the body, what we call somatic cells,
but it wasn't contributing to the germ line. And for us we wanted to go into the germ line,
because then, if we ever made a modification,
we could then generate as many mice as we want
with that modification simply by breeding.
But those cells didn't exist. And then, fortunately, at around 1980...late 1984
I heard rumors that Martin Evans in Cambridge, England had actually started developing
cells that may work. At that time he called them EK cells
and those cells, what he did is to isolate, rather than isolating these cells from a tumor
he isolated very similar cells from an embryo.
And simply used EC cells as the driving force to say, "What kind of cells I want."
but now instead of deriving it from a tumor he was isolating them from the embryo themselves
and those cells looked like they may be capable of contributing to the germ line
and therefore would be a suitable substrate for us to do gene targeting with.
So I called up Martin Evans, this was Christmas of, now, 1985 and
asked him if I could go to his lab to learn how to work with these cells, and he was
very generous and allowed us to go. My wife and I went there,
spent several weeks learning how to work with these cells,
how to use those cells, then actually how to make embryos in a sense of introducing them
to what we call a blastocyst, a pre-implantation embryo
and then these EK cells are now called ES cells, would then contribute to the formation
of the embryo proper, once we implant it into the mouse
but fortunately now these cells were contributing to the germ line.
So they were perfect for what we wanted to do
in terms of modifying mice.
So that essentially gave you the background for us to be able to then, not only
go from directing DNA at a particular target in cell culture
but now extending it to formation of mice with specific mutations.