We're going to discuss today the definition that you two gave which
is making a genetically identical copy in terms of organisms.
And I want to begin by distinguishing with you the terms
reproductive and therapeutic cloning.
Reproductive cloning refers to
making a whole organism, doing cloning with the intent,
and here we are in our board of life, same place we were last time,
stem cells cloning, let's not dwell there. Reproductive cloning,
the stuff that covers' of Time Magazine are made of.
The outcomes of reproductive cloning, the desired outcome is that you get
the whole organism.
And I want to distinguish that from therapeutic cloning where the
desired outcome is that you get some kind of cells and specifically
stem cells. So reproductive cloning is the stuff
of movies, it's the stuff of science fiction and of people having fun
with covers of the New York Times Magazine. It's also the subject of
scams. And I'll tell you at the outset, there are no human clones
now. Human cloning has not yet worked, but I am sure someone
somewhere is trying. And we'll come back to that later
one in the lecture. The other type of cloning,
therapeutic cloning is something that extends directly from our
lecture last time. When you think about stem cells and
you think about using those in repair, one of the issues that comes
up, and this comes up in any kind of repair mechanism,
repair therapy, transplantation of any kind is the question of autology
or self or matching, whether or not the donated tissue
genetically matches your own If it doesn't your immune system is
going to try to reject it, and if it does you have a lot of
problems. The sense of therapeutic cloning is that one may be able to
take cells from an adult person and turn them into an embryo and then
use the embryo cells, I'll tell you how in a moment,
and then use the resulting embryo and the inner cell mass of the
embryo to make various stem cell lines, as we discussed
in last lecture. And the promise of therapeutic
cloning is this autologous nature to it, that you would get something
that was an exact tissue match. So let's discuss a bit more, why
one might want to do this various types of cloning.
In reproductive cloning there are some senses of replacing people who
have been lost, some way of improving or overcoming
infertility. Spare parts, this is the stuff that
some good books have been made of. House of the Scorpion from Nancy
Farmer. If any of you have read that it's directly relevant to this.
If you haven't, House of the Scorpion it's called.
It's a very interesting book that thinks about a time in our society
where we can clone exact copies of ourselves. And these copies are
made brain damages. And they are kept in hospitals and
they are used for spare parts as the real person ages.
OK? Sounds weird, sounds peculiar, sounds unethical,
but it is possible. And I suspect there are people who would be quite
keen on having this as something that could be done.
Useful producer animals, there's been a sense in the
agricultural industry that if one can make identical copies of
particular animals it would be very useful in getting whole herds of
goats, for example, that are making very high quantities
of a protein that is pharmaceutically useful,
some kind of drug for example. And there are in different ways.
That kind of approach has already taken. Therapeutic cloning,
as I said, autologous stem cells and the use, which I may or may not have
time to touch on, of these cells in correcting genes
which are mutant and which are bad. But let's step back for a moment
because the question of cloning has a long history.
And its history really started with a question that we have dwelt on for
a number of lectures, and this is the question of how cell
types are determined. And the question of when a cell type
is determined, whether or not that is accompanied
by some irrevocable change in the DNA. So this cell type
determination, or let's say differentiation because
that's the end point, as you know, is cell type
differentiation caused by DNA change?
Or let me actually say DNA change or DNA loss, which is the big one.
And this question, and you could imagine, as you form a retinal cell
or as you form a skin cell, you might get these different cell
types because you throw out, you actually delete from the genome
whole batteries of genes. OK? Or you make lots of copies of
another gene. So you may actually change the DNA.
You kind of know the answer to this question because I've already told
you that all cells have pretty much got the same amount of DNA,
the same type of DNA. And what's important is whether or not
different genes are active or not. But how did I tell you that? How
was I able to tell you that? So let's spend a few minutes
exploring that. So the first question that was
asked in terms of the cell type differentiation question was the
question of cellular potency.
Where cellular potency, and we're on number two of your
handout, where cellular potency asked in an early embryo,
when can an early embryonic cell or set of cells support growth of the
entire organism? In other words, at what time during
development is there totipotency, is there complete information in the
cell? And this is to exemplify the kind of
experiment that was done, as exemplified here in the sea
urchin where you can take an eight cell stage sea urchin embryo and
transect it in one plane or another plane. And if you do it in one
plane you get an abnormal embryo and a normal embryo.
They say abnormal but it's sort of normal-ish. And in the other plane
you get out two normal, but small, larvae. OK?
And what this said was that the cells of this eight cell embryo are
not equivalent to one another. And, indeed, even four cells put
together, if you bisect in the wrong plane, was not sufficient to confer
totipotency to those cells. So at the eight cell stage in the
sea urchin, the cells are not totipotent. And you can do similar
types of experiments in many organisms.
And what you find is that in the very early embryo,
you can often split the embryo into its component cells and you can get
out a number of genetically identical organisms.
This clearly happens in humans in the case of identical twins.
It happens in armadillos routinely where armadillos,
for their own reasons, split into eight cells at the eight
cell stage, and you get identical octuplets out of most armadillo
pregnancies. OK? So there's totipotency but it's
transient. And this transient nature of this
cellular potency, so the embryonic potency is
transient.
And the question was then raised why? Was it something that happened to
the cytoplasm or was it something that happened to the nucleus?
And this is where we get into the questions of nuclear potency and the
beginnings of cloning as we know it.
And the technique that you need to
know is called somatic cell nuclear transfer.
Abbreviated SCNT.
All right. So this is what SCNT looks like. This is number three on
your handouts. And it goes like this.
One takes an egg, early embryonic cell that you know is able to
support total embryonic development with its own nucleus,
and you go in and you remove the egg chromosomes. You can do this
because most eggs are arrested at meiotic metaphase two.
The chromosomes are condensed and they are gelatinous,
and you can actually remove them. I'll show you a movie of this in a
moment. You can remove these egg chromosomes, and you can then get a
nucleus from some adult or other cell type. And you can put it in a
glass pipette, and you can pipette it into the
enucleated egg. The egg heals up.
It's now got a substituted nucleus. And you can go and you can ask what
this egg which its somatic nucleus can do.
And the somatic cell nucleus is diploid so you don't need
fertilization because you've already got the right complement of
chromosomes. And if you do that, as was done in frogs in the early
1960s, you get results like this. The host egg came from a frog which
was brown and the donor nucleus came from a frog, or cells of a frog
which was albino. And that was important because when
you looked at all the cloned frogs they were all albinos.
OK. And so you could see that they came from the donor nuclei.
Great question.
See me afterwards. I actually don't know the answer.
That's a great question. OK. Here are the cloned frogs.
OK? And you can get buckets full of these things,
but. I've seen buckets full of these things. OK.
But. OK. But before the but. So let's do a couple of things here.
So want I can tell you is that in a 500 cell frog embryo,
one can very clearly distinguish cellular potency and nuclear potency
differences. So if you take any cells from a 500 cell frog embryo,
you isolate the single cells and culture them as single cells,
none of them will form an embryo. However, if you isolate nuclei from
each of those cells and transplant those nuclei each into an enucleated
egg, 100% of them will form normal embryos.
So there is a massive difference between cellular and nuclear potency
at that stage. It tells you that the nuclei are
totipotent and the problem was with the cytoplasm of the cell and that
the egg cytoplasm has got something in it that is special to support
development. So that was a very positive result,
OK, that you could get these cloned embryos, these cloned frogs from
nuclei that by cellular experiments were not totipotent anymore, but --
But, and this is really important, if you did that experiment with
nuclei derived from older and older embryo, here's blastula,
gastrula, neurula tail bud and so on all the way up into adult cells,
the frequency of embryos that arose from, or frogs that arose from the
somatic cell nuclear transfer plummeted.
So if you did the experiment at blastula stages you got 80%,
even 100% success rate with the cloning with your somatic cell
nuclear transfer. If you did it at gastrula it would
drop to 50%. By late gastrula, which is just in frogs, about five
hours after late blastula, you were down at around 15%.
And by the time a day later had gone by and you were a tail bud
embryo, you were close to zero percent success.
So although nuclei were totipotent at some stage,
there was a tremendous decrease in the potency of the nuclei as
development went on. And so that raised issues,
which we'll come back to in a little bit. Now, after the success with
frogs, people started looking to mammals immediately to see if they
could clone mammals. And there is a real checkered
history here and some shady science and some claims particularly that in
mice one could clone mouse embryos and could do it with
high efficiency. And it turned out that all of that
data was faked, and that really put a damper on the
field for several decades. And it wasn't until the folks in
Edinburgh in 1997 tried again and did it with an open mind that they
were able to clone this sheep, Dolly, who never knew how famous she
was, who was the first mammal who was cloned by nuclear transfer.
So how does nuclear transfer work in mammalian embryos?
The principle is identical to what I've shown you in the frogs,
but I'm going to show you a few movies to give you a sense of how
it's really done. So this is a microscope that's set
up for somatic cell nuclear transfer. And these things here are
micromanipulators, including injection and suction
devices that I'll show you in a minute.
I'm going to show you a series of three movies that were made by a
very talent MIT graduate student, Kevin Eggan who is now a fellow over
at Harvard, in Professor Rudolf Jaenisch's lab,
one of my colleagues over at the Whitehead Institute.
And the first movie is going to be the isolation of nuclei
from somatic cells.
So here is a glass pipette, and Kevin is drawing up a cell into
the pipette. And he is going to spit out, he has, you'll
see it again. He has spit out the contents of the
cell other than the nucleus. So there's this little thing in
there that's a nucleus. So here's another cell.
He's pipetting it up and down to break it open and is retaining the
nucleus because it fits well in the pipette and doesn't come out as
readily as the other cell contents. There goes the cell into the
pipette, and here comes the cell debris, and in the pipette is
retained the nucleus. OK. Step two, removing the chromosomes
from an unfertilized egg. This thing is a suction device that
sucks and holds the mouse egg. Here is a microinjection needle.
And it's not pointed because it's actually being helped by an electric
pulse to enter the egg. And Kevin is now pulling out the
metaphase plate from an egg. Those are the chromosomes from the
egg. How does he know where they are? Well, he's a smart guy.
And if you've looked at enough mouse eggs, you can actually see
them under the right microscopy conditions. OK.
So there goes the pipette again and here comes some more chromosomes.
And there you go. OK. Easy when you know how.
There. OK. So once you've got your
bucket load of eggs that are enucleated and you've got your
pipette full of somatic cell nuclei, you can put the two together. So
here's your enucleated egg held on by suction and here's your pipette
that has got those nuclei that came from the somatic cells.
Watch the barrel of the pipette or the microinjection pipette
and you will see it. There is goes.
There's a nucleus going down into the cell. And if you watch
carefully we will see when the pipette comes out.
You really have got to get it out of there. That nucleus is gone.
OK. Here's another one. There's the nucleus. You've got to watch.
There it goes. You can actually see it going it out if you look
carefully. He's really making sure he pushes it out.
And there, another nucleus somatically transferred.
OK. And the third one here is the nucleus. A little pulse of electric
current to get the pipette in, and then you push the nucleus out by
pressure, and there you are. And the last one, here's a lost
nucleus.
There is goes. All right. OK.
So this works. It does work.
These mice are living proof that somatic cell nuclear transfer gives
clones and so is Dolly, but there are issues. There are
many, many problems.
Cloning by somatic cell nuclear transfer is very inefficient.
It depends somewhat on the species
one has looked at, but it is generally around or less
than 1%. So fewer than one in a hundred of those mouse eggs actually
make it on to give you a mouse. The ones that do,
although they might look normal, turn out all to have something wrong
with them. So in mice 100% of the animals that you get out have got
something wrong with them. And this is probably true in all
clones that have been made. It was true of Dolly who died an
early death due to arthritis and various other issues.
And it's true in essentially every animal, in all animals that have
been carefully examined. So what are the problems?
This is to remind you again that cloning efficiency from adult nuclei
is very low. One of the problems, and these animals would not be
viable, is something called large offspring syndrome.
This is a normal newborn mouse and this is a cloned mouse.
And you can see that it's at least double the size of
the normal mouse. This is the placenta from the normal
mouse and from the cloned mouse also enormous. So there is an issue here
with the size of embryos or the size of the animals.
There's also an issue with lifespan. This is a survival curve for mice.
And mice cloned animals are pretty much all dead by about 750 days
after birth. Whereas, for most wild type or even animals
made by artificial reproductive technologies, your survival is way,
way longer than at. At least double that time.
So there is an issue with survival in general. And I'll show you a
little bit, I pulled out a couple of panels of some primary research data
to show you that the reason these animals are abnormal is because
their gene expression is abnormal. OK? So this is a PCR-based assay
looking at various RNAs from various different genes.
OK? These are, each of these names up here is a
gene name. It doesn't matter what they are. And each of these white
bands here is the demonstration that an RNA is present.
In a normal embryo there is a band under each of these gene names
indicating that the RNA is present. In a cloned embryo, and this is
true for all cloned embryos, you see that at least three bands
are missing. And here's one I didn't diagram,
didn't put an arrow on. Four bands are missing. And in different
cloned embryos different RNAs are missing. So you can show that in
cloned embryos where you look gene expression is abnormal.
Why is gene expression abnormal? Well, there are a couple of reasons.
One is that in fact we're wrong and that the DNA content of cells really
is changing as they get older. But that's not supported by projects
like the Human Genome Project which suggests that the genes,
in fact, are stable in all cells. A much more plausible and accepted
explanation of the data has to do with the control of gene expression.
And an aspect of the control of gene expression that we have
mentioned, although not by this particular term.
And so I want to introduce you to a new term which is epigenetics.
Epigenetics means outside genetics.
And this refers to the control of gene expression that does not
directly bear on the DNA sequence. We can have an argument about this
later, but that's how I'm going to define it now.
This refers to the regulation of gene expression --
-- that is not base
sequence directed.
That is not directed by the DNA base sequence. All right.
And I wrote it for you. Let's look at some examples.
This is a cloned cat. This is a kitten and this is its
mom. And you can see they're very cute but they are different from one
another. OK? They're different but they've got exactly the same DNA
content. They're different. They're expressing different genes.
OK? This is where we've been. Let's move somewhere else.
I would think we might have someone who is one of an identical twin pair
in the class. Anyone an identical twin?
Oh, yeah. Well, we're statistically borderline.
They're one in a thousand. So we don't have it.
Do we have fraternal twins? Are you a fraternal twin? One.
OK. All right. A few. OK. So there we're OK.
All right. OK. Identical twins.
Identical twins are generally not absolutely identical.
They might look a bit different, they might have different behaviors,
and they may have different more quantitative traits.
The term concordance refers to whether or not twins share a trait.
And that's particularly useful in looking to see whether diseases are
causes by DNA based mutations. There's something called
scleroderma which is a skin disorder that has 98% concordance.
If one twin has it the other twin has it. And it is directed by
specific changes in the DNA sequence. On the other hand,
asthma only has a 54% concordance. And that suggests that asthma is
directed, this is a high concordance, it's higher than fraternal twins
have, but it certainly doesn't reach the heights of scleroderma.
And what the data suggests is that scleroderma is caused only due to
changes in DNA base sequence and asthma is caused due to changes in
the DNA base sequence as well as something else,
and the something else is believed to be changes in gene expression
that are regulated above the level, at a different level than the base
sequence per se. Now, if we think back to molecular
biology and the regulation of transcription,
I pulled this slide from one of your previous handouts,
your gene, your promoter and your promoter activity leading to
production of RNA. That is the level of gene
expression that seems to go wrong in cloned embryos.
We talked about regulation of transcription by binding of
transcription factors to the promoter.
And you've just had an exam on this previously. And also by something
called chromatin modification. And epigenetics has to do with
chromatin. Again, this is an old slide.
You've had it previously. And I pointed out to you that DNA
is packaged into tightly wound coils called chromatin by winding around
particular proteins. And the particular proteins it winds
around specifically are called histones, and these histones and the
DNA wound around them are inhibitory to transcription and they must be
loosened up or removed to allow transcription to take place.
So bear that in mind while I tell you something else that can happen
to DNA. DNA can be covalently modified, and this is on your
handout. This is number six on your handout. DNA can be covalently
modified. In particular,
cytosine, the base cytosine can have a methyl group added to its 5
position, and it is now called 5-methylcytosine.
And this does not change the base sequence of the DNA,
but it does have a profound affect on whether or not the DNA is
transcriptionally active. So let me go through a cartoon with
you to indicate how this is, and then I'll come back to the
relevance of this for cloned animals and their abnormalities.
So this is number seven on your handout.
The DNA that I've shown in black, and I've shown double-stranded DNA
as a single line here so don't get confused, is wound around purple
barrels of histone proteins. And this chromatin configuration
where the DNA is wound around histone proteins has to be removed
or has to be modified to activate transcription.
When the histones are removed the transcription factors can do their
thing and you can get transcription taking place.
And it turns out that when the DNA is methylated that does not happen.
Methylation prevents the removal of the histones from the DNA template,
from the gene, and thereby prevents transcription from taking place from
a gene. OK? So on the handout that I've posted on the Web,
I have put an explanation of why methylation prevents
histone removal. I'm not going to go through it in
class, but you can go and look at it for your interest on the Web posted.
OK? But what I want you to have in your minds now is that methylation
prevents DNA removal. Now, methylation therefore,
because it can repress transcription, is used by this body to use by the
cell as a mechanism to regulate which genes are active
and which cell type. So in cell type one where there's a
gene that I have depicted as so, double-stranded and there's a
promoter, no methylation, it's expressed. That same gene in
cell type two might be methylated and therefore not expressed.
This is number nine on your handout. Reciprocally gene two might be
methylated in cell type one where it's not expressed,
not methylated in cell type two where it is expressed and so on.
And what you can get from this is that in a particular cell type a set
of genes will have a characteristic methylation pattern.
And you can get a kind of methylation profile of the entire
genome that dictates which genes are going to be active and which are not
active, or is one level of regulation that dictates which genes
are active and expressed and which are not.
There is another level of methylation that has to do with
what's in the egg and the sperm and what methylation patterns are there.
And it turns out that not only are there cell type specific methylation
differences but there are gender specific differences.
And the methylation patterns of female genes, of genes in the female
are different than some of the methylation patterns of genes in the
male. This particular kind of methylation difference is called
imprinting. Here's an example. Here's a gene.
Gene one in the egg has a particular methylation pattern.
That same gene two, gene one in the sperm has a different methylation
pattern. OK? And this difference makes it a so-called imprinted gene.
Here's gene two. Also differences between the male and the female
copies of the genes. And here's gene three which has the
same methylation pattern in both egg and sperm and is therefore
not imprinted. So there are these two kinds of
methylation that take place, the cell type specific and the
gender specific. And these all get put together into
a complicated arrangement of changing methylation patterns during
development. And I'm going to go through this. This is number ten on
your handout. And I'm going to go through this as an animated cartoon.
So here is, represented by the colors, the methylation pattern in
the egg and in the sperm. They are imprinted with their own
methylation patterns. During fertilization they join to
form a zygote which goes on to make an embryo. Now,
early on during embryogenesis some cells are set aside.
And we looked at this with determinants in Caenorhabditis.
Some cells are set aside to make the future germ cells.
And very interestingly as that happens there is a demethylation of
all the imprinting of methyl groups.
And as those germ cells then go onto become egg or sperm new methylation
takes place and you get the imprinted patterns put back on egg
and sperm appropriately. It's a complicated process.
And if you think about it a little you'll see there are some real
issues there, but I'm throwing this out as a cartoon.
Also in the embryo new methylation patterns arise that I've called
embryonic methylation patterns. So there are enzymes that are adding
these methyl groups onto cytosine, and there are a bunch of different
enzymes. And in the embryo there are new methylation patterns that
arise. And as the embryo goes through its thing and makes its
different cell types, additional new methylation patterns
arise. I've called these somatic methylation patterns.
And they are different in the different cell types, as
I've just shown you. So there is this complicated way of
changing methylation patterns during development, and this has
implications for the chromatin structure and it regulates the
expression of genes in different cell types. It is one important
level of gene regulation. And for cloning purposes you should
know that adult methylation patterns never normally revert to
embryonic patterns. The germ cells are set aside very
early during embryogenesis, and once that has happened you go on
and make all these new methylation patterns and you never turn them
back. OK. So what does this have to do with cloning?
Well, the problem with clones and the problem with gene expression in
clones, it is believed, is that there is a problem with
changing the methylation patterns and the chromatin structure back to
the embryonic structure. So in normal developments,
I've summarized the cartoon that I just showed you in complicated form,
you move from some kind of egg-sperm methylation state that moves on,
that allows you to move on to activate the early embryonic genes.
And because you have put in the appropriate embryonic methylation
patterns. And then in a stepwise way you go on to act two,
form the adult methylation patterns and activate the adult genes.
OK? So there's a connection between the methylation patterns and correct
gene expressions. This is a summary of the big
cartoon I just showed you. After somatic cell nuclear transfer
there are three possible outcomes with regard to methylation patterns
and with the prognosis for the health of the resulting embryo.
One outcome is that this adult donor nucleus that has its adult
methylation patterns, when injected into enucleated egg,
will undergo some kind of reprogramming and will land up with
a normal embryonic set of methylation patterns.
If it does that it will be able to support normal development.
However, it is believed that this very, very rarely, probably
never happens. What is much more likely is that
this adult donor nucleus, when injected into an enucleated egg,
goes on and is partially reprogrammed by the enzymes that are
present in the egg. And it goes on to set up some kind
of abnormal set of methylation patterns, some of which look like
the embryonic patterns, some of which look like the adult
patterns. And it goes on correspondeningly to activate gene
expression abnormally. And, therefore,
to make an abnormal embryo. And then a third outcome is that
you just don't reprogram this nucleus at all,
and it's an adult nucleus sitting in the egg. And in that case you get
no embryo resulting from the somatic cell nuclear transfer at all.
So it is, and this is number 12 on your slide, on your handout.
So the notion now is that abnormal clones are abnormal because of an
inability to reprogram the DNA, they methylation patterns and the
chromatin structure to turn you into a normal embryo.
So let's move on to the final thing I want to discuss with you today
which are issues. Issues with somatic cell nuclear
transfer and the whole notion of cloning. And I have a list of them
here. They fall into two categories. One are the ethical issues and the
other are the methodological issues.
We raised last time the ethics of using human embryos to make human
stem cell lines. And this ethical issue is still
there. Is it acceptable to take a human embryo at the blastocystic
stage, when it's a ball of cells and to harvest it,
to kill it, to take the cells and to make stem cell lines?
Someone asked me whether you couldn't take one cell out of that
embryo and make a cell line out of that embryo and let the rest
of the embryo grow. And in theory you actually could do
that. It would be a pain in the neck, but you actually in theory
could do that. OK? But that's not a possibility
that's really being seriously entertained. This whole use of
human embryos to support actually making a whole organism has its real
issues. What if you do that and you land up with as you do in the mice
and the cows and the sheep and the everything else?
You land up with a whole bunch of abnormal human beings,
what are you going to do about this? What's the fate of them, what's
your commitment to these abnormal babies and what is the commitment of
society here? These are very big issues. Another issue,
which is a very real one, is where are the eggs that you're
going to do all this somatic cell nuclear transfer going to come from?
So there was a recent report from a Korean group who had actually made a
human stem cell line after somatic cell nuclear transfer.
They had used 650 human eggs. And they had used 16 women to
donate these 650 eggs. At the present rate of making
clones and making stem cell lines, you would probably need at least 500
to 1,000 human eggs per attempt to get autologous stem cells.
Now, I told you previously, when we talked about assisted
reproductive technology and you retrieve oocytes from the ovary and
do in vitro fertilization, you usually get about 20 oocytes
every time you try to get eggs from a donor.
That's a lot of people who have to donate these eggs.
And if you're actually going to try and grow these into babies that's a
lot of surrogate mothers. So this is an issue that has not
been resolved or even discussed really very well.
But these are the ethical issues that make these scientific issues
move into the realm of legislature. There is currently a federal ban on
using federal money to make human stem cell lines.
So you cannot use money from the National Institutes of Health to do
this. There is no actual ban on trying to clone humans per se.
There is a gridlock because it's not clear whether to separate
therapeutic and reproductive cloning from one another,
but these are issues that are very loaded.
And, as I mentioned last time, in the Massachusetts House we just
approved the use of private funding to make human stem cell lines in
Massachusetts, but not the use of federal funding.
We cannot do that. OK. Methods. To date no primate has been cloned.
There is something difficult about cloning primates.
No human, no monkey has been cloned yet. Why? What's the problem?
Not clear at this point, but I suspect those problems will be
overcome in the near future. OK. Some other thoughts.
What about these eggs? I told you the source of eggs for somatic cell
nuclear transfer in humans is a big issue. What if you could turn ES
cells into eggs? Could you treat then with something
and turn them into eggs? And that would overcome the need
for donors. Well, in fact, an experiment was published
a little while ago showing that you could take ES cells and you could
activate them in various ways. And activate expression of these
proteins that you should recognize, ZP1, ZP2 and ZP3. Remember those?
Yes? OK, the zona pellucida proteins. So that you could take
these cells and at least start turning them into eggs.
That's the sense that you might be able to do. OK.
And then finally the question of how can you reprogram these adult
nuclei to make the whole process more efficient?
And I'll end with telling you two thoughts that are being entertained
by people in the field. One of them is to take an adult
donor nucleus and to try to reprogram it into an embryonic state
sequentially. So if you take the adult donor nucleus --
Thank you. If you take the adult
donor nucleus, you inject it into an enucleated egg,
you let it partially reprogram, and then you take the nucleus out of
that partial embryo and put it into a new egg, that nucleus does much,
much better. And that's true in frogs as well.
And you can then sequentially reactivate the genetic program.
And that's actually one of the best arguments that the genetic material
really does stay the same. And it's a problem with the
chromatin structure of the DNA. And the other dream that people are
pursuing is to take these adult donor nuclei and incubate them in
some kind of reprogramming extract, some mix of proteins that will
magically change the chromatin structure of the nucleus and allow
it to revert to a normal embryonic nucleus so it an go on and do its
thing. And I am going to stop there. And thank you very much.