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We're going to discuss today the
definition that you two gave which

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is making a genetically identical
copy in terms of organisms.

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And I want to begin by
distinguishing with you the terms

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reproductive and therapeutic
cloning.

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Reproductive cloning refers to

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making a whole organism,
doing cloning with the intent,

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and here we are in our board of life,
same place we were last time,

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stem cells cloning, let's not dwell
there.  Reproductive cloning,

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the stuff that covers' of Time
Magazine are made of.

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The outcomes of reproductive cloning,
the desired outcome is that you get

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the whole organism.

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And I want to distinguish that from
therapeutic cloning where the

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desired outcome is that you get some
kind of cells and specifically

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stem cells.
So reproductive cloning is the stuff

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of movies, it's the stuff of science
fiction and of people having fun

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with covers of the New York Times
Magazine.  It's also the subject of

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scams.  And I'll tell you at the
outset, there are no human clones

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now.  Human cloning has not yet
worked, but I am sure someone

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somewhere is trying.
And we'll come back to that later

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one in the lecture.
The other type of cloning,

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therapeutic cloning is something
that extends directly from our

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lecture last time.
When you think about stem cells and

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you think about using those in
repair, one of the issues that comes

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up, and this comes up in any kind of
repair mechanism,

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repair therapy, transplantation of
any kind is the question of autology

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or self or matching,
whether or not the donated tissue

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genetically matches your own
If it doesn't your immune system is

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going to try to reject it,
and if it does you have a lot of

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problems.  The sense of therapeutic
cloning is that one may be able to

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take cells from an adult person and
turn them into an embryo and then

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use the embryo cells,
I'll tell you how in a moment,

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and then use the resulting embryo
and the inner cell mass of the

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embryo to make various stem cell
lines, as we discussed

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in last lecture.
And the promise of therapeutic

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cloning is this autologous nature to
it, that you would get something

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that was an exact tissue match.
So let's discuss a bit more, why

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one might want to do this various
types of cloning.

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In reproductive cloning there are
some senses of replacing people who

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have been lost,
some way of improving or overcoming

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infertility.
Spare parts, this is the stuff that

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some good books have been made of.
House of the Scorpion from Nancy

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Farmer.  If any of you have read
that it's directly relevant to this.

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If you haven't, House of the
Scorpion it's called.

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It's a very interesting book that
thinks about a time in our society

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where we can clone exact copies of
ourselves.  And these copies are

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made brain damages.
And they are kept in hospitals and

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they are used for spare parts as the
real person ages.

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OK?  Sounds weird,
sounds peculiar, sounds unethical,

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but it is possible.  And I suspect
there are people who would be quite

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keen on having this as something
that could be done.

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Useful producer animals,
there's been a sense in the

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agricultural industry that if one
can make identical copies of

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particular animals it would be very
useful in getting whole herds of

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goats, for example,
that are making very high quantities

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of a protein that is
pharmaceutically useful,

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some kind of drug for example.
And there are in different ways.

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That kind of approach has already
taken.  Therapeutic cloning,

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as I said, autologous stem cells and
the use, which I may or may not have

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time to touch on,
of these cells in correcting genes

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which are mutant and which are bad.
But let's step back for a moment

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because the question of cloning has
a long history.

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And its history really started with
a question that we have dwelt on for

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a number of lectures,
and this is the question of how cell

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types are determined.
And the question of when a cell type

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is determined,
whether or not that is accompanied

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by some irrevocable change in the
DNA.  So this cell type

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determination,
or let's say differentiation because

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that's the end point,
as you know, is cell type

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differentiation caused
by DNA change?

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Or let me actually say DNA change or
DNA loss, which is the big one.

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And this question, and you could
imagine, as you form a retinal cell

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or as you form a skin cell,
you might get these different cell

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types because you throw out,
you actually delete from the genome

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whole batteries of genes.
OK?  Or you make lots of copies of

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another gene.  So you may actually
change the DNA.

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You kind of know the answer to this
question because I've already told

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you that all cells have pretty much
got the same amount of DNA,

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the same type of DNA.  And what's
important is whether or not

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different genes are active or not.
But how did I tell you that?  How

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was I able to tell you that?
So let's spend a few minutes

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exploring that.
So the first question that was

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asked in terms of the cell type
differentiation question was the

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question of cellular potency.

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Where cellular potency,
and we're on number two of your

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handout, where cellular potency
asked in an early embryo,

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when can an early embryonic cell or
set of cells support growth of the

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entire organism?
In other words, at what time during

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development is there totipotency,
is there complete information in the

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cell?
And this is to exemplify the kind of

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experiment that was done,
as exemplified here in the sea

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urchin where you can take an eight
cell stage sea urchin embryo and

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transect it in one plane or another
plane.  And if you do it in one

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plane you get an abnormal embryo and
a normal embryo.

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They say abnormal but it's sort of
normal-ish.  And in the other plane

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you get out two normal, but
small, larvae.  OK?

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And what this said was that the
cells of this eight cell embryo are

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not equivalent to one another.
And, indeed, even four cells put

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together, if you bisect in the wrong
plane, was not sufficient to confer

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totipotency to those cells.
So at the eight cell stage in the

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sea urchin, the cells are not
totipotent.  And you can do similar

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types of experiments
in many organisms.

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And what you find is that in the
very early embryo,

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you can often split the embryo into
its component cells and you can get

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out a number of genetically
identical organisms.

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This clearly happens in humans in
the case of identical twins.

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It happens in armadillos routinely
where armadillos,

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for their own reasons,
split into eight cells at the eight

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cell stage, and you get identical
octuplets out of most armadillo

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pregnancies.  OK?
So there's totipotency but it's

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transient.
And this transient nature of this

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cellular potency,
so the embryonic potency is

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transient.

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And the question was then raised why?
Was it something that happened to

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the cytoplasm or was it something
that happened to the nucleus?

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And this is where we get into the
questions of nuclear potency and the

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beginnings of cloning
as we know it.

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And the technique that you need to

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know is called somatic cell
nuclear transfer.

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Abbreviated SCNT.

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All right.  So this is what SCNT
looks like.  This is number three on

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your handouts.
And it goes like this.

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One takes an egg, early embryonic
cell that you know is able to

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support total embryonic development
with its own nucleus,

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and you go in and you remove the egg
chromosomes.  You can do this

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because most eggs are arrested at
meiotic metaphase two.

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The chromosomes are condensed and
they are gelatinous,

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and you can actually remove them.
I'll show you a movie of this in a

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moment.  You can remove these egg
chromosomes, and you can then get a

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nucleus from some adult or other
cell type.  And you can put it in a

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glass pipette,
and you can pipette it into the

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enucleated egg.
The egg heals up.

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It's now got a substituted nucleus.
And you can go and you can ask what

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this egg which its somatic
nucleus can do.

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And the somatic cell nucleus is
diploid so you don't need

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fertilization because you've already
got the right complement of

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chromosomes.  And if you do that,
as was done in frogs in the early

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1960s, you get results like this.
The host egg came from a frog which

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was brown and the donor nucleus came
from a frog, or cells of a frog

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which was albino.
And that was important because when

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you looked at all the cloned frogs
they were all albinos.

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OK.  And so you could see that they
came from the donor nuclei.

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Great question.

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See me afterwards.
I actually don't know the answer.

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That's a great question.  OK.  Here
are the cloned frogs.

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OK?  And you can get buckets full
of these things,

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but.  I've seen buckets full of
these things.  OK.

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But.  OK.  But before the but.
So let's do a couple of things here.

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So want I can tell you is that in a
500 cell frog embryo,

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one can very clearly distinguish
cellular potency and nuclear potency

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differences.  So if you take any
cells from a 500 cell frog embryo,

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you isolate the single cells and
culture them as single cells,

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none of them will form an embryo.
However, if you isolate nuclei from

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each of those cells and transplant
those nuclei each into an enucleated

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egg, 100% of them will
form normal embryos.

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So there is a massive difference
between cellular and nuclear potency

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at that stage.
It tells you that the nuclei are

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totipotent and the problem was with
the cytoplasm of the cell and that

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the egg cytoplasm has got something
in it that is special to support

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development.  So that was a very
positive result,

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OK, that you could get these cloned
embryos, these cloned frogs from

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nuclei that by cellular experiments
were not totipotent anymore, but --

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But, and this is really important,
if you did that experiment with

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nuclei derived from older and older
embryo, here's blastula,

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gastrula, neurula tail bud and so on
all the way up into adult cells,

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the frequency of embryos that arose
from, or frogs that arose from the

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somatic cell nuclear
transfer plummeted.

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So if you did the experiment at
blastula stages you got 80%,

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even 100% success rate with the
cloning with your somatic cell

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nuclear transfer.
If you did it at gastrula it would

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drop to 50%.  By late gastrula,
which is just in frogs, about five

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hours after late blastula,
you were down at around 15%.

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And by the time a day later had
gone by and you were a tail bud

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embryo, you were close to
zero percent success.

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So although nuclei were totipotent
at some stage,

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there was a tremendous decrease in
the potency of the nuclei as

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development went on.
And so that raised issues,

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which we'll come back to in a little
bit.  Now, after the success with

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frogs, people started looking to
mammals immediately to see if they

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could clone mammals.
And there is a real checkered

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history here and some shady science
and some claims particularly that in

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mice one could clone mouse embryos
and could do it with

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high efficiency.
And it turned out that all of that

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data was faked,
and that really put a damper on the

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field for several decades.
And it wasn't until the folks in

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Edinburgh in 1997 tried again and
did it with an open mind that they

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were able to clone this sheep,
Dolly, who never knew how famous she

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was, who was the first mammal who
was cloned by nuclear transfer.

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So how does nuclear transfer work in
mammalian embryos?

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The principle is identical to what
I've shown you in the frogs,

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but I'm going to show you a few
movies to give you a sense of how

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it's really done.
So this is a microscope that's set

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up for somatic cell nuclear transfer.
And these things here are

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micromanipulators,
including injection and suction

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devices that I'll show
you in a minute.

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I'm going to show you a series of
three movies that were made by a

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very talent MIT graduate student,
Kevin Eggan who is now a fellow over

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at Harvard, in Professor Rudolf
Jaenisch's lab,

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one of my colleagues over at the
Whitehead Institute.

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And the first movie is going to be
the isolation of nuclei

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from somatic cells.

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So here is a glass pipette,
and Kevin is drawing up a cell into

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the pipette.  And he is going to
spit out, he has, you'll

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see it again.
He has spit out the contents of the

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cell other than the nucleus.
So there's this little thing in

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there that's a nucleus.
So here's another cell.

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He's pipetting it up and down to
break it open and is retaining the

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nucleus because it fits well in the
pipette and doesn't come out as

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readily as the other cell contents.
There goes the cell into the

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pipette, and here comes the cell
debris, and in the pipette is

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retained the nucleus.  OK.
Step two, removing the chromosomes

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from an unfertilized egg.
This thing is a suction device that

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sucks and holds the mouse egg.
Here is a microinjection needle.

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And it's not pointed because it's
actually being helped by an electric

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pulse to enter the egg.
And Kevin is now pulling out the

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metaphase plate from an egg.
Those are the chromosomes from the

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egg.  How does he know where they
are?  Well, he's a smart guy.

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And if you've looked at enough
mouse eggs, you can actually see

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them under the right microscopy
conditions.  OK.

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So there goes the pipette again and
here comes some more chromosomes.

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And there you go.  OK.  Easy when
you know how.

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There.  OK.  So once you've got your

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00:17:37,000 --> 00:17:41,000
bucket load of eggs that are
enucleated and you've got your

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00:17:41,000 --> 00:17:45,000
pipette full of somatic cell nuclei,
you can put the two together.  So

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00:17:45,000 --> 00:17:49,000
here's your enucleated egg held on
by suction and here's your pipette

225
00:17:49,000 --> 00:17:53,000
that has got those nuclei that came
from the somatic cells.

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00:17:53,000 --> 00:17:57,000
Watch the barrel of the pipette or
the microinjection pipette

227
00:17:57,000 --> 00:18:02,000
and you will see it.
There is goes.

228
00:18:02,000 --> 00:18:06,000
There's a nucleus going down into
the cell.  And if you watch

229
00:18:06,000 --> 00:18:10,000
carefully we will see when the
pipette comes out.

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00:18:10,000 --> 00:18:15,000
You really have got to get it out
of there.  That nucleus is gone.

231
00:18:15,000 --> 00:18:19,000
OK.  Here's another one.  There's
the nucleus.  You've got to watch.

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00:18:19,000 --> 00:18:23,000
There it goes.  You can actually
see it going it out if you look

233
00:18:23,000 --> 00:18:28,000
carefully.  He's really making sure
he pushes it out.

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00:18:28,000 --> 00:18:33,000
And there, another nucleus
somatically transferred.

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00:18:33,000 --> 00:18:38,000
OK.  And the third one here is the
nucleus.  A little pulse of electric

236
00:18:38,000 --> 00:18:43,000
current to get the pipette in,
and then you push the nucleus out by

237
00:18:43,000 --> 00:18:49,000
pressure, and there you are.
And the last one, here's a lost

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00:18:49,000 --> 00:19:03,000
nucleus.

239
00:19:03,000 --> 00:19:09,000
There is goes.
All right.  OK.

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00:19:09,000 --> 00:19:18,000
So this works.  It does work.

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00:19:18,000 --> 00:19:22,000
These mice are living proof that
somatic cell nuclear transfer gives

242
00:19:22,000 --> 00:19:26,000
clones and so is Dolly,
but there are issues.  There are

243
00:19:26,000 --> 00:19:35,000
many, many problems.

244
00:19:35,000 --> 00:19:39,000
Cloning by somatic cell nuclear
transfer is very inefficient.

245
00:19:39,000 --> 00:19:48,000
It depends somewhat on the species

246
00:19:48,000 --> 00:19:52,000
one has looked at,
but it is generally around or less

247
00:19:52,000 --> 00:19:57,000
than 1%.  So fewer than one in a
hundred of those mouse eggs actually

248
00:19:57,000 --> 00:20:02,000
make it on to give you a mouse.
The ones that do,

249
00:20:02,000 --> 00:20:08,000
although they might look normal,
turn out all to have something wrong

250
00:20:08,000 --> 00:20:13,000
with them.  So in mice 100% of the
animals that you get out have got

251
00:20:13,000 --> 00:20:19,000
something wrong with them.
And this is probably true in all

252
00:20:19,000 --> 00:20:24,000
clones that have been made.
It was true of Dolly who died an

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00:20:24,000 --> 00:20:30,000
early death due to arthritis and
various other issues.

254
00:20:30,000 --> 00:20:34,000
And it's true in essentially every
animal, in all animals that have

255
00:20:34,000 --> 00:20:38,000
been carefully examined.
So what are the problems?

256
00:20:38,000 --> 00:20:42,000
This is to remind you again that
cloning efficiency from adult nuclei

257
00:20:42,000 --> 00:20:47,000
is very low.  One of the problems,
and these animals would not be

258
00:20:47,000 --> 00:20:51,000
viable, is something called large
offspring syndrome.

259
00:20:51,000 --> 00:20:55,000
This is a normal newborn mouse and
this is a cloned mouse.

260
00:20:55,000 --> 00:20:59,000
And you can see that it's at least
double the size of

261
00:20:59,000 --> 00:21:04,000
the normal mouse.
This is the placenta from the normal

262
00:21:04,000 --> 00:21:08,000
mouse and from the cloned mouse also
enormous.  So there is an issue here

263
00:21:08,000 --> 00:21:13,000
with the size of embryos or the size
of the animals.

264
00:21:13,000 --> 00:21:17,000
There's also an issue with lifespan.
This is a survival curve for mice.

265
00:21:17,000 --> 00:21:22,000
And mice cloned animals are pretty
much all dead by about 750 days

266
00:21:22,000 --> 00:21:27,000
after birth.  Whereas,
for most wild type or even animals

267
00:21:27,000 --> 00:21:31,000
made by artificial reproductive
technologies, your survival is way,

268
00:21:31,000 --> 00:21:36,000
way longer than at.
At least double that time.

269
00:21:36,000 --> 00:21:41,000
So there is an issue with survival
in general.  And I'll show you a

270
00:21:41,000 --> 00:21:47,000
little bit, I pulled out a couple of
panels of some primary research data

271
00:21:47,000 --> 00:21:52,000
to show you that the reason these
animals are abnormal is because

272
00:21:52,000 --> 00:21:57,000
their gene expression is abnormal.
OK?  So this is a PCR-based assay

273
00:21:57,000 --> 00:22:03,000
looking at various RNAs from various
different genes.

274
00:22:03,000 --> 00:22:07,000
OK?  These are,
each of these names up here is a

275
00:22:07,000 --> 00:22:11,000
gene name.  It doesn't matter what
they are.  And each of these white

276
00:22:11,000 --> 00:22:15,000
bands here is the demonstration that
an RNA is present.

277
00:22:15,000 --> 00:22:19,000
In a normal embryo there is a band
under each of these gene names

278
00:22:19,000 --> 00:22:23,000
indicating that the RNA is present.
In a cloned embryo, and this is

279
00:22:23,000 --> 00:22:27,000
true for all cloned embryos,
you see that at least three bands

280
00:22:27,000 --> 00:22:31,000
are missing.
And here's one I didn't diagram,

281
00:22:31,000 --> 00:22:35,000
didn't put an arrow on.  Four bands
are missing.  And in different

282
00:22:35,000 --> 00:22:40,000
cloned embryos different RNAs are
missing.  So you can show that in

283
00:22:40,000 --> 00:22:45,000
cloned embryos where you look gene
expression is abnormal.

284
00:22:45,000 --> 00:22:49,000
Why is gene expression abnormal?
Well, there are a couple of reasons.

285
00:22:49,000 --> 00:22:54,000
One is that in fact we're wrong and
that the DNA content of cells really

286
00:22:54,000 --> 00:23:00,000
is changing as they get older.
But that's not supported by projects

287
00:23:00,000 --> 00:23:06,000
like the Human Genome Project which
suggests that the genes,

288
00:23:06,000 --> 00:23:12,000
in fact, are stable in all cells.
A much more plausible and accepted

289
00:23:12,000 --> 00:23:19,000
explanation of the data has to do
with the control of gene expression.

290
00:23:19,000 --> 00:23:25,000
And an aspect of the control of
gene expression that we have

291
00:23:25,000 --> 00:23:31,000
mentioned, although not by this
particular term.

292
00:23:31,000 --> 00:23:38,000
And so I want to introduce you to a
new term which is epigenetics.

293
00:23:38,000 --> 00:23:44,000
Epigenetics means outside genetics.

294
00:23:44,000 --> 00:23:46,000
And this refers to the control of
gene expression that does not

295
00:23:46,000 --> 00:23:49,000
directly bear on the DNA sequence.
We can have an argument about this

296
00:23:49,000 --> 00:23:51,000
later, but that's how I'm going to
define it now.

297
00:23:51,000 --> 00:23:54,000
This refers to the regulation of
gene expression --

298
00:23:54,000 --> 00:24:03,000
-- that is not base

299
00:24:03,000 --> 00:24:17,000
sequence directed.

300
00:24:17,000 --> 00:24:23,000
That is not directed by the DNA base
sequence. All right.

301
00:24:23,000 --> 00:24:29,000
And I wrote it for you.
Let's look at some examples.

302
00:24:29,000 --> 00:24:33,000
This is a cloned cat.
This is a kitten and this is its

303
00:24:33,000 --> 00:24:37,000
mom.  And you can see they're very
cute but they are different from one

304
00:24:37,000 --> 00:24:42,000
another.  OK?  They're different but
they've got exactly the same DNA

305
00:24:42,000 --> 00:24:46,000
content.  They're different.
They're expressing different genes.

306
00:24:46,000 --> 00:24:51,000
OK?  This is where we've been.
Let's move somewhere else.

307
00:24:51,000 --> 00:24:55,000
I would think we might have someone
who is one of an identical twin pair

308
00:24:55,000 --> 00:25:00,000
in the class.  Anyone
an identical twin?

309
00:25:00,000 --> 00:25:03,000
Oh, yeah.  Well,
we're statistically borderline.

310
00:25:03,000 --> 00:25:07,000
They're one in a thousand.  So we
don't have it.

311
00:25:07,000 --> 00:25:11,000
Do we have fraternal twins?
Are you a fraternal twin?  One.

312
00:25:11,000 --> 00:25:15,000
OK.  All right.  A few.  OK.  So
there we're OK.

313
00:25:15,000 --> 00:25:19,000
All right.  OK.
Identical twins.

314
00:25:19,000 --> 00:25:23,000
Identical twins are generally not
absolutely identical.

315
00:25:23,000 --> 00:25:27,000
They might look a bit different,
they might have different behaviors,

316
00:25:27,000 --> 00:25:31,000
and they may have different more
quantitative traits.

317
00:25:31,000 --> 00:25:35,000
The term concordance refers to
whether or not twins share a trait.

318
00:25:35,000 --> 00:25:39,000
And that's particularly useful in
looking to see whether diseases are

319
00:25:39,000 --> 00:25:44,000
causes by DNA based mutations.
There's something called

320
00:25:44,000 --> 00:25:48,000
scleroderma which is a skin disorder
that has 98% concordance.

321
00:25:48,000 --> 00:25:53,000
If one twin has it the other twin
has it.  And it is directed by

322
00:25:53,000 --> 00:25:57,000
specific changes in the DNA sequence.
On the other hand,

323
00:25:57,000 --> 00:26:02,000
asthma only has a 54% concordance.
And that suggests that asthma is

324
00:26:02,000 --> 00:26:06,000
directed, this is a high concordance,
it's higher than fraternal twins

325
00:26:06,000 --> 00:26:10,000
have, but it certainly doesn't reach
the heights of scleroderma.

326
00:26:10,000 --> 00:26:14,000
And what the data suggests is that
scleroderma is caused only due to

327
00:26:14,000 --> 00:26:19,000
changes in DNA base sequence and
asthma is caused due to changes in

328
00:26:19,000 --> 00:26:23,000
the DNA base sequence as well as
something else,

329
00:26:23,000 --> 00:26:27,000
and the something else is believed
to be changes in gene expression

330
00:26:27,000 --> 00:26:31,000
that are regulated above the level,
at a different level than the base

331
00:26:31,000 --> 00:26:35,000
sequence per se.
Now, if we think back to molecular

332
00:26:35,000 --> 00:26:39,000
biology and the regulation of
transcription,

333
00:26:39,000 --> 00:26:42,000
I pulled this slide from one of your
previous handouts,

334
00:26:42,000 --> 00:26:46,000
your gene, your promoter and your
promoter activity leading to

335
00:26:46,000 --> 00:26:50,000
production of RNA.
That is the level of gene

336
00:26:50,000 --> 00:26:53,000
expression that seems to go wrong in
cloned embryos.

337
00:26:53,000 --> 00:26:57,000
We talked about regulation of
transcription by binding of

338
00:26:57,000 --> 00:27:01,000
transcription factors
to the promoter.

339
00:27:01,000 --> 00:27:06,000
And you've just had an exam on this
previously.  And also by something

340
00:27:06,000 --> 00:27:11,000
called chromatin modification.
And epigenetics has to do with

341
00:27:11,000 --> 00:27:16,000
chromatin.  Again,
this is an old slide.

342
00:27:16,000 --> 00:27:21,000
You've had it previously.
And I pointed out to you that DNA

343
00:27:21,000 --> 00:27:26,000
is packaged into tightly wound coils
called chromatin by winding around

344
00:27:26,000 --> 00:27:31,000
particular proteins.
And the particular proteins it winds

345
00:27:31,000 --> 00:27:35,000
around specifically are called
histones, and these histones and the

346
00:27:35,000 --> 00:27:39,000
DNA wound around them are inhibitory
to transcription and they must be

347
00:27:39,000 --> 00:27:43,000
loosened up or removed to allow
transcription to take place.

348
00:27:43,000 --> 00:27:47,000
So bear that in mind while I tell
you something else that can happen

349
00:27:47,000 --> 00:27:51,000
to DNA.  DNA can be covalently
modified, and this is on your

350
00:27:51,000 --> 00:27:55,000
handout.  This is number six on your
handout.  DNA can be covalently

351
00:27:55,000 --> 00:27:59,000
modified.
In particular,

352
00:27:59,000 --> 00:28:03,000
cytosine, the base cytosine can have
a methyl group added to its 5

353
00:28:03,000 --> 00:28:07,000
position, and it is now called
5-methylcytosine.

354
00:28:07,000 --> 00:28:11,000
And this does not change the base
sequence of the DNA,

355
00:28:11,000 --> 00:28:14,000
but it does have a profound affect
on whether or not the DNA is

356
00:28:14,000 --> 00:28:18,000
transcriptionally active.
So let me go through a cartoon with

357
00:28:18,000 --> 00:28:22,000
you to indicate how this is,
and then I'll come back to the

358
00:28:22,000 --> 00:28:26,000
relevance of this for cloned animals
and their abnormalities.

359
00:28:26,000 --> 00:28:30,000
So this is number seven on your
handout.

360
00:28:30,000 --> 00:28:34,000
The DNA that I've shown in black,
and I've shown double-stranded DNA

361
00:28:34,000 --> 00:28:38,000
as a single line here so don't get
confused, is wound around purple

362
00:28:38,000 --> 00:28:42,000
barrels of histone proteins.
And this chromatin configuration

363
00:28:42,000 --> 00:28:46,000
where the DNA is wound around
histone proteins has to be removed

364
00:28:46,000 --> 00:28:50,000
or has to be modified to activate
transcription.

365
00:28:50,000 --> 00:28:54,000
When the histones are removed the
transcription factors can do their

366
00:28:54,000 --> 00:28:58,000
thing and you can get transcription
taking place.

367
00:28:58,000 --> 00:29:04,000
And it turns out that when the DNA
is methylated that does not happen.

368
00:29:04,000 --> 00:29:10,000
Methylation prevents the removal of
the histones from the DNA template,

369
00:29:10,000 --> 00:29:16,000
from the gene, and thereby prevents
transcription from taking place from

370
00:29:16,000 --> 00:29:22,000
a gene.  OK?  So on the handout that
I've posted on the Web,

371
00:29:22,000 --> 00:29:28,000
I have put an explanation of why
methylation prevents

372
00:29:28,000 --> 00:29:34,000
histone removal.
I'm not going to go through it in

373
00:29:34,000 --> 00:29:38,000
class, but you can go and look at it
for your interest on the Web posted.

374
00:29:38,000 --> 00:29:43,000
OK?  But what I want you to have in
your minds now is that methylation

375
00:29:43,000 --> 00:29:48,000
prevents DNA removal.
Now, methylation therefore,

376
00:29:48,000 --> 00:29:52,000
because it can repress transcription,
is used by this body to use by the

377
00:29:52,000 --> 00:29:57,000
cell as a mechanism to regulate
which genes are active

378
00:29:57,000 --> 00:30:02,000
and which cell type.
So in cell type one where there's a

379
00:30:02,000 --> 00:30:06,000
gene that I have depicted as so,
double-stranded and there's a

380
00:30:06,000 --> 00:30:11,000
promoter, no methylation,
it's expressed.  That same gene in

381
00:30:11,000 --> 00:30:16,000
cell type two might be methylated
and therefore not expressed.

382
00:30:16,000 --> 00:30:20,000
This is number nine on your handout.
Reciprocally gene two might be

383
00:30:20,000 --> 00:30:25,000
methylated in cell type one where
it's not expressed,

384
00:30:25,000 --> 00:30:30,000
not methylated in cell type two
where it is expressed and so on.

385
00:30:30,000 --> 00:30:34,000
And what you can get from this is
that in a particular cell type a set

386
00:30:34,000 --> 00:30:39,000
of genes will have a characteristic
methylation pattern.

387
00:30:39,000 --> 00:30:43,000
And you can get a kind of
methylation profile of the entire

388
00:30:43,000 --> 00:30:48,000
genome that dictates which genes are
going to be active and which are not

389
00:30:48,000 --> 00:30:53,000
active, or is one level of
regulation that dictates which genes

390
00:30:53,000 --> 00:30:58,000
are active and expressed
and which are not.

391
00:30:58,000 --> 00:31:02,000
There is another level of
methylation that has to do with

392
00:31:02,000 --> 00:31:06,000
what's in the egg and the sperm and
what methylation patterns are there.

393
00:31:06,000 --> 00:31:10,000
And it turns out that not only are
there cell type specific methylation

394
00:31:10,000 --> 00:31:14,000
differences but there are gender
specific differences.

395
00:31:14,000 --> 00:31:18,000
And the methylation patterns of
female genes, of genes in the female

396
00:31:18,000 --> 00:31:22,000
are different than some of the
methylation patterns of genes in the

397
00:31:22,000 --> 00:31:26,000
male.  This particular kind of
methylation difference is called

398
00:31:26,000 --> 00:31:31,000
imprinting.  Here's an example.
Here's a gene.

399
00:31:31,000 --> 00:31:35,000
Gene one in the egg has a
particular methylation pattern.

400
00:31:35,000 --> 00:31:40,000
That same gene two, gene one in the
sperm has a different methylation

401
00:31:40,000 --> 00:31:44,000
pattern.  OK?  And this difference
makes it a so-called imprinted gene.

402
00:31:44,000 --> 00:31:49,000
Here's gene two.  Also differences
between the male and the female

403
00:31:49,000 --> 00:31:54,000
copies of the genes.
And here's gene three which has the

404
00:31:54,000 --> 00:31:58,000
same methylation pattern in both egg
and sperm and is therefore

405
00:31:58,000 --> 00:32:03,000
not imprinted.
So there are these two kinds of

406
00:32:03,000 --> 00:32:07,000
methylation that take place,
the cell type specific and the

407
00:32:07,000 --> 00:32:12,000
gender specific.
And these all get put together into

408
00:32:12,000 --> 00:32:16,000
a complicated arrangement of
changing methylation patterns during

409
00:32:16,000 --> 00:32:21,000
development.  And I'm going to go
through this.  This is number ten on

410
00:32:21,000 --> 00:32:26,000
your handout.  And I'm going to go
through this as an animated cartoon.

411
00:32:26,000 --> 00:32:30,000
So here is, represented by the
colors, the methylation pattern in

412
00:32:30,000 --> 00:32:35,000
the egg and in the sperm.
They are imprinted with their own

413
00:32:35,000 --> 00:32:39,000
methylation patterns.
During fertilization they join to

414
00:32:39,000 --> 00:32:43,000
form a zygote which goes on to make
an embryo.  Now,

415
00:32:43,000 --> 00:32:47,000
early on during embryogenesis some
cells are set aside.

416
00:32:47,000 --> 00:32:51,000
And we looked at this with
determinants in Caenorhabditis.

417
00:32:51,000 --> 00:32:55,000
Some cells are set aside to make
the future germ cells.

418
00:32:55,000 --> 00:32:59,000
And very interestingly as that
happens there is a demethylation of

419
00:32:59,000 --> 00:33:04,000
all the imprinting
of methyl groups.

420
00:33:04,000 --> 00:33:08,000
And as those germ cells then go onto
become egg or sperm new methylation

421
00:33:08,000 --> 00:33:12,000
takes place and you get the
imprinted patterns put back on egg

422
00:33:12,000 --> 00:33:17,000
and sperm appropriately.
It's a complicated process.

423
00:33:17,000 --> 00:33:21,000
And if you think about it a little
you'll see there are some real

424
00:33:21,000 --> 00:33:26,000
issues there, but I'm throwing this
out as a cartoon.

425
00:33:26,000 --> 00:33:30,000
Also in the embryo new methylation
patterns arise that I've called

426
00:33:30,000 --> 00:33:34,000
embryonic methylation patterns.
So there are enzymes that are adding

427
00:33:34,000 --> 00:33:38,000
these methyl groups onto cytosine,
and there are a bunch of different

428
00:33:38,000 --> 00:33:42,000
enzymes.  And in the embryo there
are new methylation patterns that

429
00:33:42,000 --> 00:33:45,000
arise.  And as the embryo goes
through its thing and makes its

430
00:33:45,000 --> 00:33:49,000
different cell types,
additional new methylation patterns

431
00:33:49,000 --> 00:33:53,000
arise.  I've called these somatic
methylation patterns.

432
00:33:53,000 --> 00:33:57,000
And they are different in the
different cell types, as

433
00:33:57,000 --> 00:34:01,000
I've just shown you.
So there is this complicated way of

434
00:34:01,000 --> 00:34:06,000
changing methylation patterns during
development, and this has

435
00:34:06,000 --> 00:34:12,000
implications for the chromatin
structure and it regulates the

436
00:34:12,000 --> 00:34:17,000
expression of genes in different
cell types.  It is one important

437
00:34:17,000 --> 00:34:22,000
level of gene regulation.
And for cloning purposes you should

438
00:34:22,000 --> 00:34:28,000
know that adult methylation patterns
never normally revert to

439
00:34:28,000 --> 00:34:33,000
embryonic patterns.
The germ cells are set aside very

440
00:34:33,000 --> 00:34:38,000
early during embryogenesis,
and once that has happened you go on

441
00:34:38,000 --> 00:34:43,000
and make all these new methylation
patterns and you never turn them

442
00:34:43,000 --> 00:34:48,000
back.  OK.  So what does this have
to do with cloning?

443
00:34:48,000 --> 00:34:54,000
Well, the problem with clones and
the problem with gene expression in

444
00:34:54,000 --> 00:34:59,000
clones, it is believed,
is that there is a problem with

445
00:34:59,000 --> 00:35:04,000
changing the methylation patterns
and the chromatin structure back to

446
00:35:04,000 --> 00:35:09,000
the embryonic structure.
So in normal developments,

447
00:35:09,000 --> 00:35:13,000
I've summarized the cartoon that I
just showed you in complicated form,

448
00:35:13,000 --> 00:35:18,000
you move from some kind of egg-sperm
methylation state that moves on,

449
00:35:18,000 --> 00:35:23,000
that allows you to move on to
activate the early embryonic genes.

450
00:35:23,000 --> 00:35:27,000
And because you have put in the
appropriate embryonic methylation

451
00:35:27,000 --> 00:35:32,000
patterns.  And then in a stepwise
way you go on to act two,

452
00:35:32,000 --> 00:35:37,000
form the adult methylation patterns
and activate the adult genes.

453
00:35:37,000 --> 00:35:42,000
OK?  So there's a connection between
the methylation patterns and correct

454
00:35:42,000 --> 00:35:47,000
gene expressions.
This is a summary of the big

455
00:35:47,000 --> 00:35:52,000
cartoon I just showed you.
After somatic cell nuclear transfer

456
00:35:52,000 --> 00:35:57,000
there are three possible outcomes
with regard to methylation patterns

457
00:35:57,000 --> 00:36:02,000
and with the prognosis for the
health of the resulting embryo.

458
00:36:02,000 --> 00:36:07,000
One outcome is that this adult donor
nucleus that has its adult

459
00:36:07,000 --> 00:36:12,000
methylation patterns,
when injected into enucleated egg,

460
00:36:12,000 --> 00:36:17,000
will undergo some kind of
reprogramming and will land up with

461
00:36:17,000 --> 00:36:22,000
a normal embryonic set of
methylation patterns.

462
00:36:22,000 --> 00:36:27,000
If it does that it will be able to
support normal development.

463
00:36:27,000 --> 00:36:32,000
However, it is believed that this
very, very rarely, probably

464
00:36:32,000 --> 00:36:36,000
never happens.
What is much more likely is that

465
00:36:36,000 --> 00:36:40,000
this adult donor nucleus,
when injected into an enucleated egg,

466
00:36:40,000 --> 00:36:43,000
goes on and is partially
reprogrammed by the enzymes that are

467
00:36:43,000 --> 00:36:47,000
present in the egg.
And it goes on to set up some kind

468
00:36:47,000 --> 00:36:51,000
of abnormal set of methylation
patterns, some of which look like

469
00:36:51,000 --> 00:36:54,000
the embryonic patterns,
some of which look like the adult

470
00:36:54,000 --> 00:36:58,000
patterns.  And it goes on
correspondeningly to activate gene

471
00:36:58,000 --> 00:37:02,000
expression abnormally.
And, therefore,

472
00:37:02,000 --> 00:37:06,000
to make an abnormal embryo.
And then a third outcome is that

473
00:37:06,000 --> 00:37:11,000
you just don't reprogram this
nucleus at all,

474
00:37:11,000 --> 00:37:16,000
and it's an adult nucleus sitting in
the egg.  And in that case you get

475
00:37:16,000 --> 00:37:20,000
no embryo resulting from the somatic
cell nuclear transfer at all.

476
00:37:20,000 --> 00:37:25,000
So it is, and this is number 12 on
your slide, on your handout.

477
00:37:25,000 --> 00:37:29,000
So the notion now is that abnormal
clones are abnormal because of an

478
00:37:29,000 --> 00:37:34,000
inability to reprogram the DNA,
they methylation patterns and the

479
00:37:34,000 --> 00:37:39,000
chromatin structure to turn you into
a normal embryo.

480
00:37:39,000 --> 00:37:44,000
So let's move on to the final thing
I want to discuss with you today

481
00:37:44,000 --> 00:37:49,000
which are issues.
Issues with somatic cell nuclear

482
00:37:49,000 --> 00:37:55,000
transfer and the whole notion of
cloning.  And I have a list of them

483
00:37:55,000 --> 00:38:00,000
here.  They fall into two categories.
One are the ethical issues and the

484
00:38:00,000 --> 00:38:06,000
other are the methodological
issues.

485
00:38:06,000 --> 00:38:09,000
We raised last time the ethics of
using human embryos to make human

486
00:38:09,000 --> 00:38:13,000
stem cell lines.
And this ethical issue is still

487
00:38:13,000 --> 00:38:17,000
there.  Is it acceptable to take a
human embryo at the blastocystic

488
00:38:17,000 --> 00:38:21,000
stage, when it's a ball of cells and
to harvest it,

489
00:38:21,000 --> 00:38:25,000
to kill it, to take the cells and to
make stem cell lines?

490
00:38:25,000 --> 00:38:29,000
Someone asked me whether you
couldn't take one cell out of that

491
00:38:29,000 --> 00:38:33,000
embryo and make a cell line out of
that embryo and let the rest

492
00:38:33,000 --> 00:38:37,000
of the embryo grow.
And in theory you actually could do

493
00:38:37,000 --> 00:38:41,000
that.  It would be a pain in the
neck, but you actually in theory

494
00:38:41,000 --> 00:38:45,000
could do that.
OK?  But that's not a possibility

495
00:38:45,000 --> 00:38:49,000
that's really being seriously
entertained.  This whole use of

496
00:38:49,000 --> 00:38:53,000
human embryos to support actually
making a whole organism has its real

497
00:38:53,000 --> 00:38:57,000
issues.  What if you do that and you
land up with as you do in the mice

498
00:38:57,000 --> 00:39:02,000
and the cows and the sheep and the
everything else?

499
00:39:02,000 --> 00:39:06,000
You land up with a whole bunch of
abnormal human beings,

500
00:39:06,000 --> 00:39:10,000
what are you going to do about this?
What's the fate of them, what's

501
00:39:10,000 --> 00:39:14,000
your commitment to these abnormal
babies and what is the commitment of

502
00:39:14,000 --> 00:39:18,000
society here?  These are very big
issues.  Another issue,

503
00:39:18,000 --> 00:39:22,000
which is a very real one,
is where are the eggs that you're

504
00:39:22,000 --> 00:39:26,000
going to do all this somatic cell
nuclear transfer going to come from?

505
00:39:26,000 --> 00:39:30,000
So there was a recent report from a
Korean group who had actually made a

506
00:39:30,000 --> 00:39:34,000
human stem cell line after somatic
cell nuclear transfer.

507
00:39:34,000 --> 00:39:38,000
They had used 650 human eggs.
And they had used 16 women to

508
00:39:38,000 --> 00:39:43,000
donate these 650 eggs.
At the present rate of making

509
00:39:43,000 --> 00:39:48,000
clones and making stem cell lines,
you would probably need at least 500

510
00:39:48,000 --> 00:39:53,000
to 1,000 human eggs per attempt to
get autologous stem cells.

511
00:39:53,000 --> 00:39:57,000
Now, I told you previously,
when we talked about assisted

512
00:39:57,000 --> 00:40:02,000
reproductive technology and you
retrieve oocytes from the ovary and

513
00:40:02,000 --> 00:40:07,000
do in vitro fertilization,
you usually get about 20 oocytes

514
00:40:07,000 --> 00:40:12,000
every time you try to get
eggs from a donor.

515
00:40:12,000 --> 00:40:16,000
That's a lot of people who have to
donate these eggs.

516
00:40:16,000 --> 00:40:20,000
And if you're actually going to try
and grow these into babies that's a

517
00:40:20,000 --> 00:40:24,000
lot of surrogate mothers.
So this is an issue that has not

518
00:40:24,000 --> 00:40:28,000
been resolved or even discussed
really very well.

519
00:40:28,000 --> 00:40:32,000
But these are the ethical issues
that make these scientific issues

520
00:40:32,000 --> 00:40:36,000
move into the realm of legislature.
There is currently a federal ban on

521
00:40:36,000 --> 00:40:40,000
using federal money to make human
stem cell lines.

522
00:40:40,000 --> 00:40:44,000
So you cannot use money from the
National Institutes of Health to do

523
00:40:44,000 --> 00:40:48,000
this.  There is no actual ban on
trying to clone humans per se.

524
00:40:48,000 --> 00:40:52,000
There is a gridlock because it's
not clear whether to separate

525
00:40:52,000 --> 00:40:56,000
therapeutic and reproductive cloning
from one another,

526
00:40:56,000 --> 00:41:01,000
but these are issues that are very
loaded.

527
00:41:01,000 --> 00:41:05,000
And, as I mentioned last time,
in the Massachusetts House we just

528
00:41:05,000 --> 00:41:09,000
approved the use of private funding
to make human stem cell lines in

529
00:41:09,000 --> 00:41:13,000
Massachusetts,
but not the use of federal funding.

530
00:41:13,000 --> 00:41:18,000
We cannot do that.  OK.  Methods.
To date no primate has been cloned.

531
00:41:18,000 --> 00:41:22,000
There is something difficult about
cloning primates.

532
00:41:22,000 --> 00:41:26,000
No human, no monkey has been cloned
yet.  Why?  What's the problem?

533
00:41:26,000 --> 00:41:30,000
Not clear at this point, but I
suspect those problems will be

534
00:41:30,000 --> 00:41:35,000
overcome in the near future.
OK.  Some other thoughts.

535
00:41:35,000 --> 00:41:39,000
What about these eggs?  I told you
the source of eggs for somatic cell

536
00:41:39,000 --> 00:41:43,000
nuclear transfer in humans is a big
issue.  What if you could turn ES

537
00:41:43,000 --> 00:41:47,000
cells into eggs?
Could you treat then with something

538
00:41:47,000 --> 00:41:51,000
and turn them into eggs?
And that would overcome the need

539
00:41:51,000 --> 00:41:55,000
for donors.  Well,
in fact, an experiment was published

540
00:41:55,000 --> 00:41:59,000
a little while ago showing that you
could take ES cells and you could

541
00:41:59,000 --> 00:42:04,000
activate them in various ways.
And activate expression of these

542
00:42:04,000 --> 00:42:09,000
proteins that you should recognize,
ZP1, ZP2 and ZP3.  Remember those?

543
00:42:09,000 --> 00:42:13,000
Yes?  OK, the zona pellucida
proteins.  So that you could take

544
00:42:13,000 --> 00:42:18,000
these cells and at least start
turning them into eggs.

545
00:42:18,000 --> 00:42:23,000
That's the sense that you might be
able to do.  OK.

546
00:42:23,000 --> 00:42:28,000
And then finally the question of
how can you reprogram these adult

547
00:42:28,000 --> 00:42:33,000
nuclei to make the whole process
more efficient?

548
00:42:33,000 --> 00:42:37,000
And I'll end with telling you two
thoughts that are being entertained

549
00:42:37,000 --> 00:42:42,000
by people in the field.
One of them is to take an adult

550
00:42:42,000 --> 00:42:46,000
donor nucleus and to try to
reprogram it into an embryonic state

551
00:42:46,000 --> 00:42:51,000
sequentially.  So if you take the
adult donor nucleus --

552
00:42:51,000 --> 00:42:59,000
Thank you.  If you take the adult

553
00:42:59,000 --> 00:43:03,000
donor nucleus,
you inject it into an enucleated egg,

554
00:43:03,000 --> 00:43:07,000
you let it partially reprogram,
and then you take the nucleus out of

555
00:43:07,000 --> 00:43:11,000
that partial embryo and put it into
a new egg, that nucleus does much,

556
00:43:11,000 --> 00:43:15,000
much better.  And that's true in
frogs as well.

557
00:43:15,000 --> 00:43:19,000
And you can then sequentially
reactivate the genetic program.

558
00:43:19,000 --> 00:43:23,000
And that's actually one of the best
arguments that the genetic material

559
00:43:23,000 --> 00:43:27,000
really does stay the same.
And it's a problem with the

560
00:43:27,000 --> 00:43:31,000
chromatin structure of the DNA.
And the other dream that people are

561
00:43:31,000 --> 00:43:35,000
pursuing is to take these adult
donor nuclei and incubate them in

562
00:43:35,000 --> 00:43:40,000
some kind of reprogramming extract,
some mix of proteins that will

563
00:43:40,000 --> 00:43:44,000
magically change the chromatin
structure of the nucleus and allow

564
00:43:44,000 --> 00:43:49,000
it to revert to a normal embryonic
nucleus so it an go on and do its

565
00:43:49,000 --> 00:43:52,000
thing.  And I am going to stop there.
And thank you very much.