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Down the Rabbit Hole: Cloning and all things Dolly the Sheep – Bill Ritchie

Bill Ritchie is a retired embryologist who carried out the cloning procedure that led to the creation of Dolly the Sheep. Starting his science career in 1972 at an agricultural research facility in Scotland, Ritchie later gained an Open University degree which ultimately led him to the Roslin Institute.

Please note the transcript has been edited for brevity and clarity.

FLG: Hello everyone and hello Bill thank you for joining me today as we go down the rabbit hole and take a look at some of the niche aspects of genomics. Today, we’re going to be talking about all things cloning and of course, Dolly the sheep, which Bill was involved in. But before we start, Bill if you could just introduce yourself and give us a little bit about your background.

Bill: My name is Bill Ritchie. I probably came to science by a slightly convoluted route. I did a very practical course in agriculture. I then had the opportunity to do an Open University course, and at that time I worked for what was the Animal Breeding Research Organisation, which developed eventually into the Roslin Institute. When my job on the farm (the Stanhope farm down in the Scottish Borders) finished, I moved to Roslin where I did large animal anaesthesia for a few years. Then, because the equipment became available and I showed an interest, I started micromanipulating embryos – and it’s developed from there. I did eventually get my PhD by publication and that was actually in Hungary. Recently, I became a Fellow of the Royal Society of Biology. So, quite an interesting route into science, and that was certainly the way that things developed. Because I had lots of these skills, I was probably the ideal candidate for doing micromanipulation. A little bit of engineering skills, a little bit of science – the two came together – as well as good hand-eye coordination through the various sports that I played in the past.

FLG: Would you be able to provide us with a brief history of cloning?

Bill: When I started and when I got the equipment it was very basic. I took over from a PhD student and that equipment came available. The real problems at that time were that there were no good culture methods for sheep embryos. You could culture mouse or those sorts of embryos – the culture for these was very well worked out. But in sheep, we could only culture to very basic levels. The other real problem was that the glass micro tools, which were used to micro manipulate these embryos, just were not available commercially. So, the idea was that I had to first learn how to use tools to actually make the very small glass micro tools before I could actually even do any micromanipulation. And all these things took at least a day out of the week to make any basic culture medium, to make the tools. Another big problem at that time was that there was no way of in vivo maturation of oocytes. So, the very material that we were making had to be recovered from live animals, and that involves superovulation. This work obviously went on during the winter when sheep are actually mating, so we could only work during the winter months. And we could hopefully get a reasonable number of oocytes from the animals by superovulation, and that involved some surgery as well.

FLG: Why was a sheep chosen and not a mouse?

Bill: It always seems to be counterproductive to use a fairly large animal. But there are some technical reasons. The maternal zygotic transition of the embryo (this is where the embryos starts turning on genes) occurs in the mouse at the two-cell stage and occurs in the sheep somewhere around the eight-cell stage. So, it was thought that perhaps we would have that little extra bit of time when the oocyte or embryo was more malleable, and we could actually use that to be more successful. It proved that we could use embryos, and then cells to a much degree later on, but certainly in those early days that was one of the reasons. The other reason was that we knew how to work with these animals – we had worked with sheep for a long time. A lot of the experiments we had carried out were on sheep embryos, so if we wanted to do anything useful with the embryos in the future then we had to work with a larger animal than the mouse. It’s not easy taking things like milk samples from mice (although we can do it). If you’re going to do something on a commercial basis, say you want to take milk for instance, then you would probably need to use something bigger than the mouse.

FLG: What are some of the challenges working with sheep?

Bill: The mouse oocyte is somewhere around 90 microns whereas the sheep oocyte is a little bit bigger maybe up to 150 microns, so there is a technical reason there. In the mouse, the oocyte is much more difficult to work with. The zona pellucida is very flexible in the mouse, so it is actually difficult to get through the zona pellucida. And people used various mechanical means to actually get through the zona pellucida and mouse cloning actually took off a few years after Dolly. So, there are technical reasons, as well as size (the reasons that I’ve already talked about). So, there are lots of reasons. And of course, once you’ve actually done the cloning work, you can stick it outside in a field somewhere and it looks after itself. Whereas mice are a little bit more difficult to look after – you need specialist facilities for the mice (and not so much for the sheep).

I mean physically moving sheep is a of course one problem. And because we were superovulating these animals, they had to be caught on a regular basis and given injections. At that time, the hormones that we were using had a very short half-life and it meant that the animals had to be injected twice per day with these hormones. So, actually catching and handling animals was one of the problems. Also, I’ll say this all the way through any interview – you’re only as good in these experiments as the weakest link. The person who, perhaps, is not doing their job would be a problem in any part of these experiments. So, it was very much collaborative work between people producing cells, the glass dishes had to be washed and sterilised and all sorts of things that people really don’t think of. It was a really really difficult experiment to actually carry out, certainly the Dolly one.

FLG: Megan and Morag were the first mammals to be successfully cloned from cultured cells. There was also Polly and Molly. Can you share a brief background about how these experiments paved the way to clone Dolly the Sheep?

Bill: My boss (Keith Campbell) always said that Morag and Megan were the enabling technologies to actually allow the cloning to progress and for things to actually work. The reason for that was that the first animals that certainly I made (there were quite a number of animals made prior to Dolly) but the technique of course was to take an embryo which had developed to maybe the eight-cell stage, so the cells are fairly easily disaggregated. So, you can take one big cell containing a nucleus and place it into an enucleated oocyte (an oocyte where you have removed the DNA). And at this stage, one of the problems is that you can’t see it, it’s not bound by a nucleus in the oocyte. So, removing it is one of the big problems. But you then had perhaps 8-16 cells that you could easily disaggregate and actually use for cloning. Cloning has a success rate of somewhere around 10%. So, even if you had 16 cells, the possibility of making more than one clone is very very small. So, the animals prior to Morag and Megan were made by this method. Whereas Morag and Megan were made from cultured cells. So, the fact that you have cultured cells means you have many many cells, literally millions of cells. So, you can think about being able to genetically modify those cells, you can actually have a cell line of cells which are all genetically identical. So, you can make a number of animals which are all going to be the same, which are all going to produce the same proteins that you’re looking at. That’s really the big breakthrough – in being able to go from a very small number of identical cells to millions of cells, and potentially being able to genetically modify those cells and produce animals which could have an additional gene at that time or even knockout a gene – and we did both of those in subsequent years.

FLG: What was the difference between the clones?

Bill: We used a cultured inner cell mass from a blastocyst to produce the first animals from cultured cells. Then, the question is, how do you develop that? Can you use foetal cells for instance? And it developed from there. Although its generally not recognised, we actually used three different cell lines for the Dolly idea. We had some very early cells in development, some from a foetus and then we had the adult cells. The fact that we were interested in using those techniques to make animals which had an added human protein – these were all steps in the development to actually do that.

FLG: How did Polly and Molly differ to Dolly? And why did it not receive as much attention?

Bill: I think that the idea of Dolly really just caught the imagination of not only scientists, but really the whole world, and people then had the idea well maybe we can clone ourselves. Maybe it was one of those things that people in the back of their mind thought perhaps this is just one of those things that they can connect with science, and they can see something in their life that perhaps would be affected by it.

FLG: What was it like at the time when the news broke?

Bill: The Morag and Megan paper was around 25 years ago, and the problem was, it was dominated by other stories in the press at the time, so it really didn’t reach its full potential. But when the Dolly paper was meant to be published, the story was broken early by some of the Sunday newspapers. And that, of course, caused problems. We weren’t really ready at the time to get things working. The press were not going to be there for 2 or 3 days after the paper had been produced. But because the story had been broken early, all hell had broken loose well before the time we expected it to be, and not that we really anticipated it would be such a big story. I was beginning to get phone calls from my boss, Sir Ian Wilmut, on the Saturday evening that the story was going to be broken early on the Sunday (Sunday newspapers knew about it). So, he had already started fielding calls from other people and was going to go into the Roslin Institute to start dealing with some of the calls. I think he did a small number perhaps on the Sunday. But by the Monday, all hell had broken loose! I was still busy doing cloning work at the time, and I entered the Institute, and the car park was full of vans with dishes on the roof, just trying to get a story. Many of these people had already flown in from the United States and all around the world – it was bedlam!

FLG: Did you have any people protesting outside?

Bill: No, not for some time. People did eventually catch on. At that time, there were some problems certainly in England, certainly roundabout Cambridge and Oxford, with protestors. But we didn’t have a huge amount of problem with them – I think it was too far for them to travel! We did have them on occasions.

FLG: I wasn’t born when Dolly the sheep first got cloned and so many animals have been cloned since, including a black-footed ferret last year. But everyone still talks about Dolly – why was the impact of Dolly so significant?

Bill: I mean it is interesting isn’t it that people remember this one scientific experiment. In actual fact, it was rated higher than the first Mars landing in that year, just because of its significance in human medicine, stem cells and how things have developed in treatment for diseases. But also, just the fact that we had changed a scientific law. When I was at agricultural college, the law was that once a cell had been differentiated, it couldn’t be undifferentiated. I can remember speaking to lots of people who taught science and them saying ‘Oh, it’s terrible I am going to have to change all of my lecture notes because what I have been teaching people all of my life has changed.’ And it was that sort of change. And I think it was the fact that so many people did see it. I think if it would have been a mouse, then no one would have paid a terrific amount of attention. I think what happened too, was that one of my colleagues joked about where the cells came from. And calling her Dolly also helped the process of making it very memorable for people who maybe knew about Dolly Parton and connected the two. Dolly Parton herself knows about it and I think is fairly happy with the fact she has been named after it!

FLG: Many question the purpose of cloning – for example, cats have been cloned – what is the purpose of this?

Bill: We have home office regulations in this country, so the chances of actually cloning something in a commercial basis are fairly slim. It’s not something that we would perhaps do very much in this country. People have cloned all sorts of pets. People always think that if they clone a particular animal then the subsequent clone will be a clone of that animal. Unfortunately, what they don’t realise is that the original animal and cloned animal can be really different because they are exposed to different diseases, different stimuli throughout their lives. If they are treated differently, then they are going to be totally different. The way that animals develop are more on the environment than the actual genetics, and many people don’t really understand that.

FLG: What are some of the useful applications of cloning?

Bill: The original idea was to make animals which had a human protein. At that time 25 years ago, the problem was that proteins were often extracted from blood, things like blood factors for haemophiliacs. And the problem was that it was very difficult to detect viruses in the blood, so blood products were contaminated with HIV and other viral diseases. Sheep don’t get these diseases. The idea of one of the commercial sponsors, PPL Therapeutics, was to actually make proteins from the milk of sheep. That was superseded by other methods, but this showed that it could be done – that we could actually get a flock of sheep which were identical where we could use milk and the protein could then be purified and then the person would not suffer from the same viral diseases that we had done in the past.

FLG: What about going forward, in terms organ transplantation?

Bill: Recently we’ve had some animals which were chimaeras made with human cells and primate cells. And this seems to always comes from China, but these experiments were done indeed in the early 2000s – very similar experiments. The problem with trying to make organs is what you can do is take an embryo, knockout a particular gene so perhaps you produce an animal which has a liver or pancreas that doesn’t work. Then you could add a human cell and the human cell (which has that particular gene in it) would then make a pancreas and it would be a human pancreas in that particular animal. The problem is of course, although it makes the pancreas, all the other connective tissue and blood vessels and everything still come from the animal that you transferred the cells to.  Although it all seems a good idea, the problem is that you still have a pancreas or a liver or whatever which is not completely from the cells that you want. This has been an idea for a long time, although it seems to have raised its head recently, it’s not a new idea. The problems probably still exist.

FLG: What about the applications of cloning in conservation?

Bill: At one time, I said that one of the animals we could clone would be the highland wildcat. It is probably one of the few animals that would be very easy to clone because they hybridise with domestic cats, so the oocytes from the domestic cats could be used for the wildcat. So, you could actually produce animals that way – it would be expensive, but it would be a way of increasing it. The problem with many of the other species is that we don’t know enough about the culture conditions for oocytes and embryos. We don’t have the ability at the moment to harvest oocytes to do this and we don’t have the facility to actually transfer them (unless they are an animal which is very similar like the wildcat to another species). So, these things have happened in the past – the wild sheep was cloned a long time ago and a few other animals. The problem is getting access to suitable oocytes and suitable recipient animals, especially when there are very few (unless as I said there is a domestic species which is close enough to use oocytes and surrogates for that). You know, no zoo really wants you to take an animal away and say ‘We will try and transfer an embryo into this animal and see what happens’.

FLG: Aside from the obvious ethical issues, why is cloning humans so difficult?

Bill: It’s one of those things I indicated earlier – unless you actually know a little bit about the culture or how to actually do things, then it can be difficult. We do know quite a bit about human IVF. The problem seems to be that, for some reason that we don’t understand, the success rate is very small.  Experiments have gone on in human embryos for many years and people have managed to work with them, but again, the supply of oocytes and embryos is very difficult to get. Also, human reproduction is pretty inefficient if you compare it with animals. Most wild animals have a period where they are receptive to fertilisation, so within that period, something like 95% of sheep will get pregnant. In humans, we have no actual period where it is easy to detect whether the female is receptive. We have these other technical reasons that we really don’t understand, so it’s going to be pretty difficult to clone a human. Apart from ethical reasons, we perhaps have too many people in the world as it is. I think it’s just going to be very difficult to get that number of oocytes, embryos and recipients. And if we are not 100% sure that the baby being produced is going to be fit and healthy, then that’s another reason for not doing it.

FLG: How do you think newer technologies, such as CRISPR, will improve our cloning capabilities?

Bill: It’s like everything else – science never stands still. The development of CRISPR-Cas9 is probably just one of the breakthroughs in science where instead of having to actually insert a gene and clone from it, we can perhaps have a much more targeted approach to genetic modification or genetic changes in embryos. It’s not something I know a huge amount about. I did most of my cloning work quarter a century ago, so it’s quite difficult to keep up with everything.

FLG: I know you are retired Bill, but how do you think the cloning field will evolve over the coming years?

Bill: I think unless there’s a big breakthrough in the success rate, I think it’s going to retain its scientific side of things rather than making lots of pets for people who’ve got too much money and don’t really understand what they’re doing. There have been some sniffer dogs which have been cloned, and this seems to be a trait which is actually passed on through the cloning process. But from the point of view of cloning wild animals, it is a scientific technique more than anything else and there may be developments in the future. I used to work with some of the zoos and one of the things that I said at the time was that we now have stem cells. If we can differentiate these stem cells into oocytes and sperm, then all we need to make an animal would be a sample of tissue. And from that the developments of making stem cells from any tissue more or less, then if you can make an oocyte and a sperm from there, then all you need is a surrogate to actually produce another animal. It is quite a thought really – that in the future you may only need a tissue sample to actually make another animal.

FLG: The cloning field is amazing, and I think everyone’s attention will be on it as it evolves. Thank you so much for joining me today, it has been really great talking to you, it has been really interesting!

Bill: Thank you, it is nice to speak about some of things I’ve done in the past and let’s look forward to seeing what happens in the future and how the developments of 25 years ago are going ahead and helping to treat human diseases.

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