Cloning Dolly, How and Why?
Transcript Part 2: Cell Cycle Phases
I spent a lot of time looking at the cell cycle phases of my donor and recipient. Now during a single cell cycle a cell has to do a lot of things. It basically has to double everything that it contains within itself, so that it forms two daughter cells, equal to each other and--normally in a growing cell--equal to the cell before it started.
This shows a mesophyte to oocyte and early embryos where there is no net growth, so we see a reduction in size. But what we have to consider is what is going on in the nucleus. The nucleus contains this genetic information, this DNA. That has to be duplicated, so there are two copies of all of it, and then equally segregated to these daughter cells, so they both have a full complement of the instructions. That is done in a discrete period of the cell cycle called s-phase.
Now across here I have shown the cell cycle stage of our recipient cell. The cytoplasm contains protein kinases and other proteins, which can affect these cell cycle progressions..
Down the side, I have shown the cell cycle phase of our donor cell. Now I don't want to go into this in a lot of detail because it is about an hours' lecture all on its own. What I have shown here in green are the combinations that will work, in red the combinations that won't work, and in orange the combinations that we have not really been able to do because they are very difficult to prove that you have actually done that.
So actually the combination of where this donor cell is during its growth cycle, and where this recipient cell is when the fusion occurs, can affect the DNA ploidy of the resultant blastomeres. Also, if we have inappropriate mixing, we can get a lot of DNA damage.
Having said that, I just want you to note things in here. If we are going to use these metaphase 2 oocytes, which we all claimed we were using, we should only be using cells which are in this early portion of the cell cycle or G1. We shouldn't be using these G2's and we shouldn't be using these S. So we shouldn't use cells that are replicating their DNA, and we shouldn't use cells which have already replicated their DNA.
However, what if we just activate this, so it thinks it has been fertilized, and then fuse at a later stage? You can see that we can transfer our S or our G2 phase nuclei in. Now this gives us a whole number of ways of doing the technique of nuclear transfer using our metaphase 2 oocyte. We can fuse our G1 nuclei at the time of activation. We can activate and we can fuse nuclei in from G1S or G2 stages at a later date, and hopefully they all have an equal chance of developing..
Another way we came up with, because we felt that actually exposing this DNA to the components within the oocyte may be beneficial for development, was to fuse a G1 nuclei on, leave the DNA in that environment for a while, and then to activate later.
That is all very well and good. We can work out a system where we can avoid DNA damage and avoid uncoordinated DNA replication. From a lot of other work we also began to realize that there were specific times in the life cycle of the donor cell, where it appeared we could get improved development of our reconstructed embryo. It was very interesting that this appears to be as the cells are in late G2, in mitosis or very early G1. I
In late G2, the cells are condensing all their DNA into chromosomes so they can, through a process called mitosis, be segregated into two daughter cells. In mitosis they are obviously in their chromosomes, and in G1 they are still condensed and they start de-condensing.
At this point, this change in the chromatin--this condensation of the chromatin--throws off lots of factors that are stuck on this DNA, [and] are controlling the expression of the genes within that DNA. This may be very relevant to the fact that these cell cycle stages appeared to be much more able to support development. I will come back to that a little bit later. So we learned a lot of this very early on, by doing very simple experiments using synchronized well, they weren't very simple--but by using early embryos as donors of our genetic material.
Early Developmental Biology:
Now, just a little bit of early developmental biology. As I said, during early development we have a reduction and division, and slowly we go from one cell to two cells, 4...8...16...32...and then we start going through this differentiation process..
This is just a schematic, a cartoon of the blastocyte you saw in the video earlier. During these early developmental stages, these instructions, which are in the nuclei within these cells, have very little to do with development. Most of this early development is controlled by proteins and messenger RNAs, which are inherited in this oocyte from the mother--maternally inherited proteins in messenger RNA.
As they go through development, the instructions within the nucleus have to kick in and start taking over development. This onset or increase of zygotic transcription, or from the zygotic genome the stage at which it occurs, is species dependent. So, we have a decrease in maternal control and then we have to have a pattern of gene expression which is controlled in both the spatial and temporal manner..
In the very early stages of nuclear transfer, if you actually looked at when the MZT(?), or when this zygotic transcription started to happen in early embryos, and then looked at the stage in which we could get development in nuclear transfer embryos, there was quite a nice correlation. In the mass we were really limited, and this is going back about 8 or 9 years. We could use 2 cell embryos to donate the nuclear material, but the zygotic genome was taking over at the 1-2 cell stage.
Sheep and cattle are about 8-16 cells, and we could use later stages. Then we got to this experiment of John Gurdon's, where in the frog, maternal zygotic transcription starts occurring when there are 4,000 cells, and they could use these differentiated intestinal epithelial cells..
There were a number of explanations for this. People said, "As soon as we go through the MZT, the cells are becoming more differentiated and we can't take these instructions back, or reprogram them." An alternative explanation was, in those species in which the zygotic genome has to start controlling development to the latest stage when you do nuclear transfer, the transferred chromatin has to go through more mitotic divisions or more condensations, and then releases, so it is throwing off factors that are stuck to the DNA before it has to do anything. So that may be what allows it to become progressively more reprogrammed. They are probably both true, but nowadays we know we can take cells from an adult and do nuclear transfer.
In mammals we are originally restricted to these early developmental stages and using blastomeres in these early embryos. And development was related to the stage--to the species when the stage of development was species related--and seems to be related to this onset of zygotic transcription. So, if [we] wanted to do cloning and multiply off embryos, we were really limited by the number of cells that would work and also by the efficiency of the whole process..
A lot of companies set up to try, and thought, "This is a good idea. We can get lots more quality calves by taking early embryos and splitting them up and cloning them." However, all of those companies effectively went bust, because this is a very inefficient process..
Nuclear Transfer from Somatic Cells in Sheep:
The real question was, could we do nuclear transfer, could we make animals from somatic cells, from differentiated cellular material? They had made this frog from the intestinal epithelial cell, why couldn't we do it in mammals? Why do we want to do it? .
To answer the original question that all these developmental biologists set out with at the end of the 19th and beginning of the 20th century, are nuclei equivalent? Do they still contain all of the instructions that the zygote contains? This would allow us to clone from adult animals and, more importantly, and I am going to come back to this in a little while--it would give us a route in farm animal species to genetically modify animals.
How should we go about this? We could just find a cell type in the body that works. In the first case, we just wanted cells that we could keep in a dish and do nuclear transfer with. So, we could look for a cell type that works. There are things called embryonic stem cells in the mass, and everybody said, "If the embryonic stem cell...you have got to find that in farm animal species, and then you will be able to do nuclear transfer." Fortunately, no one still found embryonic stem cells or isolated embryonic stem cells in farm animal species.
The other way was to actually try and do some modification of the chromatin in this donor nucleus, prior to nuclear transfer. Now when we started doing these experiments, this question was high on everybody's mind and people basically told us it was impossible and couldn't be done. So trying to get any money to do these experiments was rather difficult because people said it was an impossible experiment..."therefore we aren't going to waste our money...However, we think you should isolate embryonic stem cells, and if and when you have insolated embryonic stem cells, we will give you some money." .
So we went about the experiments in a different way. We said, "We can't actually isolate embryonic stem cells in sheep, but what we could do is culture sheep embryos in conditions where we may expect to get embryonic stem cells, and we can do nuclear transfer with the cells and see what happens.
We took a day-9 sheep embryo...and this is day-9 sheep blastocyst. Here it is, like a big hollow ball full of fluid. The cell layer on the outside is called a trophectoderm, and the ball of cells up here--the inner cell mass--goes on to produce the fetus.
We cut this bit out here, that goes on to produce the animal, and this goes on to produce the extra embryonic tissues, the placenta, etc. And we plated it into a dish with a feeder layer, and these are the cells that we got--these epithelial-looking cells. Each time, we have to split these up because they become inhibited in their growth [since] they are close together. We have to do a thing called a "passage," and we cut them out of the dish, split them up, and put them into another dish. Each time we passaged those cells, we did nuclear transfer with them. To avoid any cell cycle effects we just pre-activated our oocyte, so we didn't have to worry where they were in the cell cycles. And we didn't do very much nuclear transfer, if you look at these three columns here, but we managed to do it on the first three passages. Lo and behold, we got live lambs.
Working with sheep in Scotland is a fun business because you could look at it one way. You could only work for about 4 months a year, because they are very seasonal. So, it is a bit like MBL--you come here in the summer and do all these experiments, and we go there in the winter and do all these experiments on sheep.
February 1994 came, and we had only gotten out to passage 3, and unfortunately sheep don't become very good donors of eggs, so we had to stop doing the experiments. We cultured these cells over the summer and we got them out to passage 11 and we did the experiments again and we did significantly more nuclear transfer, and we didn't get any live offspring. Whether that was just bad luck or a bad year, we don't know. It is still questionable. However, we went back to the drawing board.
Now I explained very briefly to you the cell cycle. This is another way of showing a cell cycle as a circular event. Cells grow, divide, grow again, divide, and the cell cycle had a lot of work done on it. People have been very, very interested in the cell cycle because of cancer. Cells just grow when you get a tumor. They grow and they grow and they grow. You get a ball of cells and that is what causes the damage. So, a lot of money was spent on examining the growth cycle of cells..
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