- DOLLY, POLLY, MEAGAN, AND MORAGH:
WHAT THEY MEAN FOR GENETICS
Caird Rexroad Sr., Ph.D.
United States Department of Agriculture
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I thank you for the opportunity to be here today. I will try to keep this
brief and very visual and let you know what these cloned animals mean to
genetics because that is essentially what cloning is about.
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Figure 1.
Sheep Chromosomes
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If you look in any of a mammalian species the chromosomes are not greatly
different. They come in pairs and all ofthe chromosomes are in every cell of
the body. Chromosomes contain the genes, 50-100 thousand, that define our
physical characteristics. Figure 1 shows the chromosomes ofa sheep such as
Dolly.
Figure 2.
Cow Embryo
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Two of the things that cloning is about is how do these genes that are
common to every cell in the body produce such different tissues such as liver,
muscle cells. and neural cells. Cloning helps us understand this process
called differentiation. The other thing that cloning is about is the ability
to make genetic changes in these cells. The material that we work with are
sheep embryos. The diameter ofthis embryo is about one third the diameter of
a thin lead pencil (Fig. 2).
Figure 3.
Early Embryo
Development |
Now what cloning is about is to help us understand how this single cell
undergoes this process, shown in this cartoon (Fig.3), from a single cell to
a two cell until the time it implants in the uterus and eventually produces
mature adults such as this animal or lambs such as this one called Dolly. So
cloning gives us some clues as to how these things happen. This process called
differentiation is how genes that are common in every cell start in a single
cell and cause the organism to become nvo cells, four cells, and then eventually
a much more complex organism, such as a lamb.
Figure 4.
Dolly Experiment
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The first thing that I want to do is to describe the Dolly experiment to
you that was done in Scotland (Fig.4). The first thing that was done was the
genetic material from an unfertilized egg was removed using a glass pipette.
In Scotland one cell that had been growing in culture in the test tube, in this
case, a cell from a mammary gland ofa six year old sheep, was taken, placed in
close proximity to this egg from which the genetic material had been removed
and then by electrical shock, the genetic material that was from this cell now
becomes part of the egg. In some number of cases, you now have a new embryo. So
instead of having the type of tissue that might have been genetically determined
by the cultured cell, the genetic program starts all over again, to produce
embryos and the famous sheep that resulted in Dolly.
Figure 5.
Finding Egg DNA |
Figure 6.
Pipette Alignment
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Dolly's predecessors may really be more informative because they give us a way
to work with cultured tissues. In our lab with a cow embryo, the first thing
that we do is identify an egg's genetic material using a stain that shines
under an ultraviolet light (Fig. 5). We take a needle that is about ten
microns across and line it up with where we know the genetic material is and
insert the needle. (Fig. 6) With aspiration we remove that genetic material.
(Fig. 7) We have to prove that it is removed, so again we use the stained
material, turn on the ultraviolet light, and we can see that the genetic
material is now in this glass holding pipe. (Fig. 8) So now we have an enucleated
egg and this is the material that we started with when we are using the
cloning procedures that were developed throughout the institute.
Figure 7.
Aspirating DNA | |
Figure 8.
Verifying Removal
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Then we pick up a cultured cell. Fibroblast cells are very common cells
ofthe body that are fairly differentiated, but they contribute to things such
as bone, connective tissue, things that hold the body together. These are
picked up with the needle and then inserted back under this membrane that
covers the egg so that they are in very close proximity. (Fig.9 and Fig. 10)
Figure 9.
Fibroblast Added | |
Figure 10.
Ennucleated Embryo
Plus Fibroblast
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We then place this in a special low salt, sugar solution between electrodes.
(Fig. 11) These are stainless steel wires through which we pass a voltage of
about 150 volts for a fraction of a second. The two cells are now become one
cell, with genetic material mostly from the fibroblast that was added; the
rest of the material called the cytoplasm is from the unfertilized egg.
(Fig. 12). The embryo is now starting over. This method of building a new
embryo reprograms the genetic material.
Figure 11.
Embryo Between
Elctrodes
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Figure 12.
Shocked Embryo
Now joined
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One might think about the genetic information that is in every cell, as a
computer programer. So that one part ofthe program is specifying the
information for the cell to be a neuron or a muscle ceil or an embryo. During
cloning these differentiated cells are restored, such as the mammary gland
cell that produced Dolly, are now restored to a more primitive state. That
is, we get conversion of a differentiated cell into an embryo by nuclear
reprogramming. This is important because a lot of times the generic defects
that we see are the result ofthe errors in the genetic program. So this at
least gives us the opportunity scientifically to study some of the factors
that are important in this programming in the genes themselves. It does not
necessarily provide us answers, but gives us a first step in how we might get
those answers.
One set of cells we are using are from the Bovine, from a cow. Fetal
fibroblast cells are grown in culture dishes. One of the things that is very
nice about being able to make a new animal from cultured cells is that we can
do genetic manipulations in culture so that we can understand how genes
function, and what we can ultimately do with genes. We can add DNA to cells
growing in a culture dish that might contain a new gene, then we can select
out the cells that have taken up this particular gene. We can grow those
cells and use them to close a new animal.
Indeed for the sheep Polly that was reported in the news, this was just
exactly the procedure that was followed. A new gene for production of
pharmaceutical was introduced into her chromosomes. There are many things
that we know can be done in cell culture. We can remove parts of genes, even
the part that codes for a protein and tells where protein should be functioning,
or perhaps, modifying it and making it new in some way. We can maybe modify
the regulatory region, the part of the region that turns the gene on and off
so that we can make it function in new ways. We can add a new gene in and we
can also remove genes, genes that might be defective or genes that we are
interested in studying their functions. So now we can grow these cells in a
culture dish and then add those cell's genetic information back to embryos
and make new individuals.
So why do we want to clone? We want to clone to produce identical animals,
it reduces the number of animals that we have to use in experiments. Indeed in
farm practices, a lot of times the farmers would like to have cows that are
identical. It might make it easier to feed them and to milk them because of
their similarity. Clones would reduce the number of animals that we need to
use on research studies. We want to understand how genes are regulated; how
does one cell eventually becomes the billions of cells of very different kinds
in the body. Cloning can be used to introduce new genes. The process is called
transgenesis.
Many of us think that this cloning reported in the newspapers last year is
something new; it is not quite. The first sheep were cloned about 1984 when a
gentleman by the name of Steen Willadsen started cloning by using embryonic
nuclei rather than using a differentiated cell such as the mammary gland
cells that were used to produce Dolly. He actually started with embryonic
nuclei that did not need to be reprogrammed, ones that you would expect that
might be able to produce new embryos. Cattle clones have actually been
marketed or sold for production practices by a company called Granada Genetics
in the late 1980s and again they used the embryonic cell technology.
In 1996, Moragh and Molly were produced. These sheep were cloned from
fetal cells and these are probably the procedures that will be most often
used rather than using adult cells. We will use fetal cells to try to study
the genetic regulation of differentiation and development. Polly and Dolly
were found this last year and we can add to that Polly was cloned and included
a new gene. The reason that Polly was cloned was that if one clones animals
that have new genes and these genes are genes that work in the mammary gland,
that is produce their protein that is part of milk, then you can produce
protein pharmaceuticals in the mammary gland. In this case, these particular
sheep were done by another technology, but will be replicated by the Dolly
technology.
We want some questions answered using this technology, we want to know
what genes function in an embryo, what genes function in mature cells, and
how do these changes occur. So we use cloning to do that. We need to know if
this material from the unfertilized embryo is special and if it is special,
what kinds of proteins or materials are in that unfertilized egg that make
it able to reprogram the genes of these mature cells, such as mammary gland
cells back in to cells that can become embryos. Then we just simply need to
kmow what turns on or off the genes that are turned on during development. So
the science can be very complex through many levels, but these are some of the
hinds of questions that we are trying to answer using the cloning techniques
that were demonstrated with Dolly.
Questions:
Q: I believe that I heard that, with age, chromosomes reduce in size?
A: Yes.
Q: So that is why we can take chromosomes from a sheep that is six
years old, the chromosomes are therefore, shorter in Dolly then they
would be in a normally new born cow. Is that true?
A: That is a hypothesis that is one that is being tested right now. This
is also related to cancer and abnormal cell growth. During development, the
ends of the chromosomes are protected by a DNA material called telomerase
and there is an enzyme called telomerase, which extends the length of
chromosomes so that as you make new cells the enzymes that make new DNA
simply fall off. So the best way to protect a chromosome is to make it long.
So that is a normal part of the process and it is thought that as we age, the
ends ofthe chromosomes become shorter and shorter as the enzymes make
chromosomes happen to fall off sooner and sooner. So one of the things that
the group was doing involving the institute was to measure the length of
the ends ofthe chromosomes in Dolly. So that is a very important question.
Does this process of nuclear reprogramming also turn back on this enzyme
called telomerase and extend the length of those chromosomes.
Talking to Dr. Willadsen, I think that the initial indications are that
that part of the chromosome is not changed. Again the telomere length
question is a hypothesis, that is it is thought that this length is important
and it correlates in a number of cancers and abnormal growth conditions with
the disease or with age, but is has not really been definitively proven. So
this is an opportunity to look and see if you can get a normal age out of an
animal that has pre-naturally shortened telomerase.
Q: Had that egg that was ennuchated and saved to the fibrobtast been
fertilized before?
A: No. That really is a critical point because these studies have been done
both with unfertilized eggs and with fertilized eggs. Dr. Willadsen demonstrated
back in 1984 was a tremendous benefit from using the unfertilized eggs. There
is likely, something in the cytoplasm of unfertilized egg that helps turn
these cells around so that they can make embryos again.
Q: How did you get to start dividing into an embryo?
A: We do not really know. The ennudeated egg is are "alive" when we put
the genetic material in there. We think that it is actually associated with
the electrical shock and the changes in the membrane. There is this release of
calcium which is a mineral that is around the membrane edges at the time that
you shock it. You can take an unfertilized egg, shock it and it will go on
through the early stages of development by itself without the presence of DNA
contributed by a sperm. So it is possible with an electrical shock to induce
development.
Q: So this has exactly the same genetic material as its mother, the
embryo that was shocked?
A: That is right. If you take an unfertilized egg and shock it, it will
begin developing. They will only develop part of the way. It will have half
of the DNA that its mother does because it will be what is called haploid; the
chromosomes had already started segregating so it has gotten rid of half of
its DNA in anticipation of receiving the DNA from the sperm. So it has part of
the chromosonnal compliment that its mother had.
Q: Do you know exactly what type of fibroblast you introduced to it?
A: No. What we do is we actually collect fibroblast from fetuses so we
might breed a cow or sheep and then collect the fetus, culture those cells
and use that genetic material. The egg comes from slaughter house. The
unfertilized eggs come from a very different location then the fibroblast cells.
Indeed, we are trying to use, and they did in Scotland, genetic material from
different breeds to provide the fetus with fibroblast as opposed to the
unfertilized eggs. That way you can demonstrate which one actually produced
the genetic information.
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