Cloning Dolly, How and Why?
Transcript Part 3: Cell Cycle Phase G0-Quiescence
There is another cell cycle phase called G0, or quiescence, which has been very ignored. I was interested for two reasons. Firstly, you can make cells exit the cell cycle and go into quiescence simply by taking away one of the nutrients in the media that you culture them in. This quiescence stage has [also] been associated with this differentiation or specialization phase, and the cells exit the cell cycle, go through some sort of change, differentiate, and then can enter the cycle again and start growing.
Unfortunately, they also exit the cell cycle into G0 if they want to die by apoptosis or by necrosis. But as I said earlier, at G1 cells or at diploid cells and these quiescent cells exit in G1 and are diploid, can be used in these three methods of nuclear transfer. So I also wanted to see whether there were any differences in development from our three methods of nuclear transfer.
So we quiesce the cells. We use three different methods of nuclear transfer, pre-activating the chromatin in the cytoplasm, fusing at the same time as activation occurred, and activating and fusing at a later date.
And all you need to look at is this. You ended up with cells that ran to passage 13 and from passage 6, and we ended up with 5 live lambs. Unfortunately, 2 of those lambs died at birth and one died at about 10 days of age. The one that died at 10 days of age...it was a bit of a shame really, because unusually for Scotland it was very hot in July, in the 90s, and this poor little lamb kept panting and falling over. The vets thought it was just suffering from heat stress, so we had fans on it. It then eventually just keeled over and died. It had a hole in the heart, which may be a problem associated with nuclear transfer, we don't know.
But we ended up with 2 lambs, which grew quite healthy, called Megan and Morag. They grew up to be quite healthy sheep, and reproduced normally. So at this time we thought, "Oh well, we have cracked this." And as I said, we reported this in the newspaper and they all picked it up, but it quickly disappeared, which was quite good. Also, we published in the scientific literature, but because we said the cells were derived from an embryo, a lot of people thought we had isolated embryonic stem cells, when in fact we hadn't.
Why would quiescence actually help this nuclear transfer? During this early development we have to go through this programmed pattern of developmental changes, and this spatial and temporal pattern of gene expression from the zygotic nucleus. In our nuclear transfer embryos, we have got to stop whatever the cell was doing at the time when it is transferred back--switch off all of those functions, and somehow modify the chromatin... modify those instructions and recapitulate these series of events--to switch off transcription.
When we quiesce cells, they reduce their transcription. They basically go to sleep. They go into hibernation. They only do what they have to do to stay alive, and we also know there are changes in the chromatin structure. The chromatin also condenses. However, we were very impressed with these results.
Those two lambs are actually females. We went back and took a male embryo, we took (inaudible), we made a cell line, quiesced the cells and we made these four male lambs. We then thought. "We are really pushing our luck here." So we took a day-26 fetus, mashed up the fetus, and made a fetal fibroblast cell, quiesced the cells, did nuclear transfer, and made these two black Welsh mantin rams..
At the time we were working with a company in the vicinity called PPR Therapeutics, a pharmaceutical company, and they were working with mammary epithelial cells taken from a 6 year old ewe. We put these into quiescence, did the nuclear transfer, and lo and behold we got Dolly. There was only an election happening, and the whole world went bonkers. The first thing that people said is, "You have made all that up. That is a complete lie. It is an impossible experiment so you can't possibly have done it. What you have done is you have taken a lamb, taken some cells from it, put them in a dish and then said you have cloned it."
We got around that one scientifically. Next, people said, "This is a really nice cloned lamb, it is lovely. But I want to know how you also made it transgenic and got odd legs." This is what happens if you give a photograph to a photographer that is not really a biologist. Because this is the photograph that this was taken from, and that odd leg there actually belongs to Dolly's surrogate mother. So that was quite funny. Her leg is just there. It is actually on there...it is a five legged sheep that we have.. (laughing).
But to go back to the science, and back to our donor cell cycle stage. We can add our G0 stage into the stages I was telling you about earlier, this late G2 and an early G1 stage, and we can add our G0 stage in for the same reasons, in my opinion. There is a reduction in transcription and displacement to transcription factors. We are helping the nucleus by choosing it in one of these stages, to go back to that zygotic form and recapitulate that series of developmental events..
There is another reason why quiescence is important. Now I am not going to tell you much about this--and the scientists in the audience may be interested--it is because of nuclear structure. The chromosomes are located in the nucleus and they appear to have a precise localization within the nucleus. And what we have found is that transcriptionally active chromosomes are found in the interior of the nucleus, and those that aren't doing very much are sort of stuck around the edge, sort of thrown out of the way. And it is funny, because this organization exists from mid to late G1 through S, and through to this early or mid G2 phase. It is lost in G1, late G2, mitosis and G0 cells. So it may be another reason why those cells work better. And there it is also lost in senescent cells and more recently we can add senescence to this overall scheme, maybe for similar reasons, because people have actually produced animals by cloning from senescent cells.
Cloning Other Species:
So what about other species? We did this in the sheep. People say, "Why did you pick a sheep?" I was actually employed to clone cattle because they were economically important, but cattle in Scotland are about 1,000 pounds each, and cost a lot of bed and breakfast. The sheep in those first experiments cost me 3 pounds 50--5 dollars each. So there was an economic reason for it and the fact is that we can do cattle experiments..
However, since we cloned the sheep we have also cloned cattle. These are two of ours. This is Mr. Jefferson and actually, this is Mr. Jefferson as well, but we cloned this one a year later, so this is what you could call a trans-generational twin, I suppose..
And the famous experiments that you have heard about in the mice and Terry Wakiama is going to be here on the course tomorrow, showing people how the mice were cloned. People have cloned goats not far from here, Genzyme Transgenetic, and we cloned pigs last year. Several other groups have also cloned pigs...quite a few species have now been cloned using very, very similar techniques..
However, as I said earlier, it is not very efficient. All the way through the development process we have losses and abnormalities. We don't know [why that is].. We have a lot of work to do..
What sort of abnormalities do we have? We don't have the source that the press would like you to believe, where we have got this little creature here. Of course it wasn't instant success. We had a lot of failures to begin with (inaudible). I think the press... because we also had a lot of chickens at the Roslin Institute, they thought we were breeding new species.
You know, there have been lots of things associated with the technology, actually before the cloning, [having] to do with in vitro production of embryos, actually. And then there were stories that appeared in the American press "because Dolly is cloned she gets a bit violent and goes around attacking..." it also says in here, "she attacks children and bites them on the leg." .
However, it is an inefficient process and we do get a lot of abnormalities in the offspring. We have loses all the way through gestation. They die, and we don't know why, and we have this range of abnormalities. There is not really any pattern. You can't say every fetus that dies has the same abnormalities.
Not only that, the animals are born and they will drop dead within 2 minutes...within the first 24 hours...and within the first 6 months, and we don't know why. To give you an example of that, this is some work that I did. We started off with 2,541 fused embryos. We had 40 lambs born. Fifty-percent of them died within 24 hours, with a range of abnormalities, and then a lot more of them died within 6 months. If they make 6 months they seem to be completely normal, and so far they have normal life spans as far as we know. So, it is a very inefficient process. We have got a lot to learn. But it does work.
So why did we want to do this? Apart from answering the question of nuclear equivalence...I think we answered that question...that we could take cells from an adult and we could animals with binuclear transfer. We wanted to do genetic modification of farm animal species.
The objectives of genetic modification, or producing a stable, inheritable change in the genetic makeup of an animal. We want to be able to add genes but we also want to be able to take genes away, or to modify their activity.
I heard about transgenic animals 15 years ago. Yes, there was a method for making transgenic farm animals 15 years ago, and this was a very simple method. You took the gene of interest, and you injected it into one of the nuclei in the zygote. Then you transfer the zygote back into the surrogate mother. In the case of sheep, you then sat down for 5 months with your fingers crossed. And then all the lambs would be born. Some of them may contain the gene and some of them may even produce the protein or the product from that gene.
But it had a lot of disadvantages. Most of the animals you produced weren't transgenetic. We could only add a gene. We couldn't do any of these other manipulations. We couldn't take a gene away, or we couldn't modify the activity of a gene within the animal. If the integration of the gene into the rest of the instructions occurred at random, you got this variation in expression of the protein or the product of the gene.
Not only that, although you are injecting it into this one cell stage, the instructions didn't integrate always at that one cell stage. They may integrate into one of the cells when there were two cells, or to one of the cells when there were four cells. So, you end up with an animal, some of its cells have got this gene in, and others haven't.
You might say that is okay, but it isn't, because what you want to do is breed that animal on, and breed that genetic change into the generation. If it is a mosaic animal, and it just so happens that the cells that have the gene didn't end up in the germ line, you are not going to pass it on to the next generation. And if you wanted to produce a flock of sheep or herd of cattle, it was pretty slow because you ended up with one animal, and you had to breed more and more and more.
Advantages of Nuclear Transfer:
Now, imagine if we could do that by nuclear transfer. We could take some cells, put them in a dish in the lab, do our genetic manipulation, and use the techniques we have and just select cells that have been genetically modified. Then we could take the one cell that we know is genetically modified, go through nuclear transfer, and end up with a blue sheep. That would be quite good, really. It has a lot of advantages..
First of all, all of the animals you made would be transgenetic, because you checked the cells before [you] started making the animals. This is the efficiency in sheep if you do pro-nuclear injection--about 5% of the offspring are transgenetic.
It isn't instant. It isn't like mashed potatoes or anything. But if we were to do nuclear transfer rather than microinjection, if we want to produce a flock of sheep that are producing milk with a protein in for instance, we can do it in about 18 months by nuclear transfer. It would take us about 44 months by breeding and pro-nuclear injection and then breeding, and 33 to 78 for cattle. So it could speed up producing proteins, or whatever it is, whatever these transgenic changes are.
Now this is the experiment that actually produced Dolly for us. Another problem with making transgenes, is that when you add [the genes] in, it depends how you put this DNA together. What the effects are. What the expression level is like.
We were interested in making proteins in the milk of farm animals, and I am going to come to that--if I ever stop talking--in a while. So, we thought, "This is a shortcut. If we can get some mammary epithelial cells put them in a dish, put in the transgene, we can see if it works. We can pick out the cell that has the best transgene expression, and we can do nuclear transfer with it and make animals.".
This [only] half worked. We still can't keep the cells in a differentiated state, making milk proteins in a dish. We thought if we have got all these cells growing in the dish, we can start doing some precise genetic modification, and we can take genes away or modify them, and then do the nuclear transfer, because we can do this on millions and millions of cells in a dish.
So, nuclear transfer has a lot of benefits. It is faster, we can do precise modification, and all the animals would be transgenic. We would get instant flocks or herds. We would use fewer animals, and we might one day be able to pre-select for expressing high expressing lines..
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