Well, hello, Dolly!
Ten years ago, on February 27, 1997, the scientific journal Nature, published Ian Wilmut and Keith Campbell’s paper announcing the birth of a sheep known as Dolly. Dolly’s genetic parent was not the Scottish Blackface ewe that bore it, but a cell taken from the mammary gland of a Finn Dorset ewe.
Using a technique called
nuclear transfer, technicians in the lab, Bill Ritchie and Karen Mycock, laboriously removed the DNA from 430 ewes’ eggs, and inserted into 277 of these the genetic material from mammary epithelial cells. After tricking the reconstructed eggs to divide, and nurturing them in culture medium, 29 became blastocysts, the embryonic stage at which they could be transferred into a surrogate mother. And from one of these came Dolly. This was revolutionary because it showed in the mammal that DNA from an adult cell could once again behave like the DNA of an embryo.
It also showed what English physician William Harvey prophesized in 1651, that “all that is alive comes from the egg.” The egg has a major role in directing how the embryo develops, a characteristic that is exploited to drive development following nuclear transfer.
Dolly, named after Dolly Parton’s famous mammaries, was the catalyst for heated public debates world-wide on the benefits and pitfalls of nuclear transfer, still reverberating today. As BBC correspondent, Pallab Ghosh, reported when Dolly’s birth was announced, “ her ultimate legacy is the start of a scientific revolution.” It is the scientific ramifications from Dolly’s birth and life that are truly profound, and as yet have not fully been realised. Yet, the idea behind these nuclear transfer experiments was not to make copies of existing people as portrayed in movies, nor was it solely to generate patient-specific human embryonic stem cell lines, one touted therapeutic application.
These experiments were undertaken to investigate a basic biological conundrum: during the process of ‘differentiation’, the making of an adult cell, is genetic information lost? That is, are adult cells incapable of being turned back to a more embryonic form? Is the process of differentiation irreversible?
The History The birth of Dolly was really a culmination of a lot of scientific work. As Isaac Newton rightly reiterated: “If I have seen a little further, it is by standing on the shoulders of giants.” It goes back to the late 1800s when Weismann, Roux and Driesch, wanted to understand the process of differentiation. Differentiation is when an embryonic cell becomes
many adult cell types. One could visualize the embryonic cell being a bit like clay; it can be molded from one form to another. In contrast, fully differentiated adult cells such as nerve, bone or blood cells, have lost the ability to do many things and have only one primary job to do.
In 1928, Hans Spemann addressed these questions experimentally using salamander eggs. He showed that the nucleus (the part of the cell where our DNA is located) from a very early embryonic cell could within the egg instruct the growth and development of an organism, providing the forerunner experiment for nuclear transfer experiments today.
By 1952, Robert Briggs and Thomas King had transferred the nuclei of an embryo into northern leopard frog eggs and generated tadpoles. They noted that it didn’t work when the nucleus came from older, more ‘adult’ cells, suggesting an older genome might very well be stuck. However, John Gurdon, generated a fully mature African Xenopus frog when he injected non-embryonic nuclei into eggs that had had their own nuclei removed. This work, published in Nature in 1958, was the first to suggest that an adult genome could go backwards in its developmental journey and behave like an embryonic genome again.
While in the public arena it raised the specter of maverick scientists vigorously applying nuclear transfer to make human clones, the research continued. In 1984, James McGrath and Davor Solter developed methods for nuclear transfer in the most common of laboratory animals, the mouse. However, they were only successful transferring nuclei from early embryonic cells. It wasn’t until the birth of Dolly in 1997 that the idea of successful nuclear transfer using an adult mammalian genome was realized.
Where Are We Now? Since Dolly, the use of nuclear transfer of an adult genome has come a long way. It has been successfully applied to many different animal species, including cows, horses, cats, and dogs. Even, noble attempts to save endangered species from extinction by nuclear transfer have been tried, and in some cases resulted in live animals (wild cats, gaur and mouflon sheep). The farming and pharmaceutical industry touts nuclear transfer as a way to make animals with desirable characteristics, such as production of cattle that produce human proteins in their milk. While these feats are considerable, the overall success rate of this procedure remains poor. Indeed, it is remarkable when some nuclear transfer animals, like Dolly, grow up and even have their own offspring- they remain the exception.
Sadly, the legacy for most animals born from a nuclear transfer embryo is severe disabilities and a very short lifespan. So, can nuclear transfer work for human cells? And why would we attempt this? Dolly’s legacy includes the possibility of making patient-specific human embryonic stem cell lines. This would involve taking the DNA from an easily reached cell, such as a skin cell, from a person afflicted with a disease or ailment, and transferring this into an egg which has had its own DNA removed. If the egg can be coaxed into developing into a blastocyst, from this, human embryonic stem cells could be made that have the same DNA as the patient.
Human embryonic stem cells have the potential to generate any adult cell type. If adult cells made from embryonic stem cells, or the embryonic cells themselves, could be used for therapies, the next hurdle will be overcoming their rejection. This problem, of course, is no different than that experienced by recipients of donor organs, who take drugs to muffle their body’s immune system. However, if the human embryonic stem cells are made from nuclear transfer embryos containing a patient’s DNA, then rejection wouldn’t be a problem.
Nowadays, scientists, including Ian Wilmut, argue that this technique is more applicable to making novel human diseased cell lines, a revolutionary way of helping us to understand how diseases develop. This understanding would better enable us to combat disease progression, possibly even to develop novel therapies. Martin Pera, from the Keck School of Medicine, envisages that this “will offer a straightforward route to development of banks of embryonic stem cell lines of a desired phenotype.”
Sadly, the only report of the successful generation of patient-specific cell lines by Woo-Suk Hwang was subsequently exposed as a fraud. No one has yet really shown that a human embryo can develop far enough after nuclear transfer of an adult nuclei to generate embryonic stem cells and the jury is still out as to whether it will. Monkeys, our close relatives, have been born following this process, but only using embryonic and not adult cells for donor nuclei. Furthermore, the procedure was wildly inefficient, highlighting one of the biggest hurdles to human nuclear transfer, the very real problem of acquiring human eggs. In addition to the ethical issues surrounding egg donation, the inefficiency of the nuclear transfer process demands a very high number of eggs providing serious practical constraints.
With the development of this technique relying on scarce human eggs, researchers have suggested using non-human animal eggs to develop cell lines for scientific inquiry; cells that would not be transplanted into a patient, rather used to study disease. Interestingly, this proposition has met with strong objections perhaps more emotional than measured. In Australia, for example, the use of non-human animal eggs for nuclear transfer of human DNA was recently banned.
Gazing into a crystal ball to speculate where these scientific breakthroughs will lead us, we may witness the altering of an adult cell fate being engineered without human eggs. Although this science is in its infancy, if it works, it will be a more practical way of producing patient specific embryonic stem cells. The fruition of this, no matter how long it takes, will be another of Dolly’s remarkable legacies. Nuclear transfer that produced Dolly, is a technique that has allowed us to probe fundamental biological questions and is still critical for us to discover more of the eggs secrets; how they achieve the remarkable feat of guiding embryogenesis, and how they can subvert the fate of a stubborn adult genome.
For these reasons, Dolly was one amazing sheep! Anthropologist Sarah Franklin, attests to this in her reminiscing of meeting with Dolly: “I think what was noticeable about her was that she was such an individual -- somewhat ironically in contrast with her iconic status as a clone. She was very forceful and direct the way 'head ewes' often are, but also, if I can say it without sounding anthropomorphic, a bit coy. Hence, for example, she certainly knew the relationship between cameras and food and not just any food either. Her first foray was to your pockets, the way a dog might do. If she did not get what she wanted she would turn her head away, or even walk to the other end of the paddock as soon as she saw your camera. All the while she would be sneaking a few glances back to see if she was working her wiles on you”. It is interesting that out of all of the animal kingdom, it was the sheep, often parodied as blind followers, rather than leaders, that pioneered this scientific revolution.
Nuclear transfer is a technique in which the nucleus of a somatic cell (any cell of the body apart from the sperm or egg) is transferred into an egg that has had its original nucleus removed. The egg now has the same DNA, or genetic material, as the donor somatic cell. Given the right signals, the egg can be coaxed into developing as if it had been fertilized. The egg would divide to form two cells, then four cells, then eight cells and so on until the blastocyst is formed. Embryonic stem cells can be derived from this blastocyst to create cell lines that are genetically identical to the donor somatic cell. Nuclear transfer may also be referred to as Somatic Cell Nuclear Transfer (SCNT) in other literature.
