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How Was Dolly the Sheep Cloned?

The birth of Dolly on the 5th of July 1996 served as one of the greatest scientific achievements in the last century, though this breakthrough has also equally raised some of the most serious questions about the ethics of modern experimental biology.


To put it simply, Dolly was the first mammal in history to have been successfully cloned from an adult cell via a process known as somatic cell nuclear transfer. But what exactly do each of those jargons mean?


Let’s dive a little deeper into each of them.

What is cloning?


Cloning is the process of producing genetically identical organisms that have exactly the same DNA. DNA is a molecule composed of two chains of nucleotides that wrap around each other to form a double helix, and it encodes all genetic information of an organism. Its molecular structure was first discovered by the world-renowned Watson and Crick duo in 1953. It has regions called genes that encode proteins that govern almost all the characteristics of cells in an organism. For example, a gene may encode a protein that gives a certain eye colour.


Although the term ‘clone’ is usually associated with duplicating animals into identical copies by the general public, clones are, in fact, found everywhere in nature. Organisms that reproduce independently without the contribution of another individual, such as bacteria and plants, are cloning themselves through asexual reproduction. For example, a parent bacterial cell duplicates its DNA and divides into two genetically identical daughter cells. Meanwhile, certain insects such as aphids can also reproduce asexually. Besides that, even cutting a part of a plant and growing it is a form of cloning.


In the context of mammals, natural clones are organisms that are nearly genetically identical. They are produced by the splitting of a single, fertilised egg into two, resulting in the production of identical twins with similar genetic compositions as each other. Though, it is crucial to bear in mind that their genetic makeup differs from either of their parents.

Figure 1: Monozygotic (identical) twin formation in mammals. An egg cell is fertilised by a sperm cell, resulting in a single zygote. The zygote then splits, leading to the development of two genetically identical embryos.


In 1996, it turned out that this phenomenon can be done artificially in mammals (sheep. in the case of Dolly). Let's find out how.


Dolly and the somatic cell nuclear transfer experiment


The background

Dolly was named after the singer Dolly Parton and formed a part of an experiment series at the Roslin Institute and PPL Therapeutics. The team, led by Professor Sir Ian Wilmut, aimed to investigate how cells change during development and apply transgenics to animal subjects.


The whole purpose of Wilmut’s original work was to find a way to reduce the number of animal subjects used in future research. In particular, he wanted to figure out how to use animals, in particular livestock, to produce molecules of therapeutic interest (this will be discussed later when we talk about Polly, a sheep that was cloned after Dolly). This later turned into experimenting with how such cells could be used to clone an entirely new animal.


The experimental procedure

When it came to Dolly, she was cloned by somatic cell nuclear transfer, whereby the nucleus from a donor cell is transplanted into a recipient cell.


To start with the basics, a somatic cell is any cell of a living organism that is not a gamete (i.e. sperm or egg cell). Examples of this would be skin or nerve cells. Moving forward, each adult somatic cell is genetically identical to one another, meaning that they contain the same chromosomes (and therefore the same DNA too). However, it is crucial to note that this does not mean that they are physiologically similar. Why? The answer would be due to the difference in gene expressions in these various types of cells, thereby resulting in their differentiation to perform specialised roles.


Returning to the context of somatic cell nuclear transfer - as the nucleus of a donor cell (which contains the organism’s genetic material) gets transferred into a recipient, this means that the recipient organism will be genetically identical to the donor.


In essence, we can separate the somatic cell nuclear transfer into six main steps:


1. Obtaining donor cells

Donor cells are obtained from a “target” organism. In Dolly’s case, the somatic cell that she was cloned from was the mammary gland of a six-year-old Finn Dorset sheep. These donor cells were then cultured under specific conditions.


2. Fusion with an enucleated cell

The extracted donor cells were then fused with a recipient, which was an enucleated egg cell (i.e. an egg that has had its nucleus removed). For Dolly, the egg cell came from a pregnant Scottish Blackface sheep.


Essentially, it was crucial that the recipient egg is capable of supporting the embryo’s development. And since the enucleated egg was taken from a pregnant sheep, it was able to do its job.


3. Epigenetic reprogramming

Now that this cell contains the donor’s genetic material, the genetic material can be reprogrammed using a process that erases all prevailing expression tags from the genome (because remember - this is the genetic material of a somatic cell, not an embryo). As a result, this ensures a variable and adaptive genetic expression as the embryo develops.


For Dolly’s case, artificial agents were used so that the expression pattern from the mammary gland cell was reverted back to the embryonic state. Thus, the cell could develop into all tissues that are essential to produce the sheep. As a result, these all lead to the production of a zygote (a fertilised egg).


4. In vitro development

The zygote is allowed to divide and develop until the blastocyst stage in vitro (meaning in a test tube or glass) over a time period of six to seven days.


5. Embryo implantation

At this point, the embryo can be implanted into a surrogate mother.


6. Embryonic development and birth

As time goes by, the embryo develops normally into a fetus within the uterus of the surrogate mother until it is finally born.

Figure 2: Somatic cell nuclear transfer that resulted in the cloning of Dolly the sheep. A somatic cell was taken from the mammary gland of a six-year-old Finn Dorset sheep and cultured in a low-nutrient medium. This arrests the cell cycle. At the same time, an egg cell was extracted from a Scottish Blackface sheep that was 3.5-months pregnant. The nucleus was then removed by micro-pipetting, thereby producing an enucleated egg. Following that, the somatic donor cell was fused with the enucleated egg by electric pulses. This ended up with the production of a fertilised egg (zygote). The zygote, in the lab, was allowed to develop for six to seven days into an embryo. After that, the embryo was finally implanted into the surrogate mother and allowed to develop. This figure was adapted from Wells (2010).


The biography of Dolly

Throughout her life, Dolly managed to give birth to six lambs, thereby proving that genetic clones are fertile and are able to produce offspring.


She lived to the age of six (which is a relatively young age for a sheep) before eventually dying of a respiratory illness. In particular, Dolly became infected with a virus known as the Jaagsiekte sheep retrovirus (JSRV) together with other sheep at the Roslin Institute. This virus basically caused a particular type of lung cancer in sheep.


Furthermore, a number of genetic complications surfaced when Dolly was one-year-old. To elaborate, she had telomeres that were much shorter in comparison to other sheep that were of her age. Telomeres refer to the caps located at the ends of chromosomes, whereby their function is to protect the genes contained in the chromosomes. And as the organism age, the telomeres will start to degrade, thereby causing the DNA to get damaged over time. So, in other words, Dolly appeared to be far older (genetically) than expected. Thus, this could have been another contributing factor to her relatively early death.


Nonetheless, even with shortened telomeres, Dolly (at the age of one) did not exhibit any conditions that were associated with premature aging. Hence, there was insufficient evidence demonstrating that Dolly’s death was directly caused by the cloning procedure.


What happened next?


Cloning became a controversial topic

Dolly marked an important milestone in scientific development as she was the first mammal cloned from an adult cell.


However, when she was first announced to the world in 1997, she caused quite a stir in the world of politics, religion, as well as the broader scientific community. Many governments acted quickly to criminalise human cloning and established committees that could advise on the ethics of cloning. Besides that, the news about Dolly had also caused a great amount of panic amongst quite a few religious leaders, whereby the Vatican had even released a statement arguing human cloning should be banned in order to maintain the dignity of human life.


Fast forward to our world today, the Human Reproductive Cloning Act of 2001 states that it is illegal to implant an embryo that has not been created through fertilisation into a woman in the UK. In short, human cloning remains a controversial and complex topic for a multitude of reasons.


Polly the Sheep came into existence

Shortly after Dolly, the same research group produced another sheep whom they named Polly.


However, Polly was not only a clone. She was also genetically modified, which makes her arguably a greater scientific feat than Dolly (but she received far less media attention). In particular, Polly was modified to carry a gene encoding a protein known as Factor IX (Factor 9), which is involved in blood clotting.


People who are deficient in Factor IX have a disease called Hemophilia B Leyden, whereby their blood is unable to clot properly. Hence, transgenic animals like Polly are of great interest as they have the ability to produce therapeutic compounds/molecules that may be a suitable treatment option. Therefore, this demonstrates how cloning could be an alternative way to produce therapeutic compounds as opposed to other methods such as through bacterial expression systems (as bacteria is often unable to support the production of human proteins due to its sheer complexity).


Why should we care?


Cloning has a lot of useful (but also questionable) applications

The ability to clone entire animals from somatic cells has multiple potential uses.


As mentioned before, transgenic animals could theoretically be used to produce therapeutic compounds at a cheaper rate. Furthermore, this could allow the rapid production of desired livestock possessing advantageous characteristics. For example, we could choose to clone a disease-resistant phenotype, which will limit the spread of disease and hence unnecessary culling procedures in the near future.


Cloning could also be used in conservation efforts in order to preserve endangered species, whereby we could freeze and store somatic and egg cells of endangered animals and reintroduce them back into their habitats in the future if necessary.


Moreover, we could potentially apply cloning during the process of xenotransplantation (i.e. the cross-species transplantation of tissues and organs). Specifically, there has been some hope that cloning transgenic pigs via somatic cell nuclear transfer so that they can be used as immunologically compatible organ donors or disease models.


Even with all these potential applications, the question still remains - is it ethical to use cloning in such a way for our own benefit?


Should human cloning even cross our minds?

To put it simply, human cloning remains controversial, with many arguments for and against the process.


Overall, the arguments against human cloning greatly outweigh those supporting the idea, but it is still interesting to understand the points raised by both ends of the debate.


Arguments for human cloning

Firstly, cloning could offer a treatment for infertility, whereby a heterosexual couple could use the egg cell from the female partner and a somatic cell from the male partner to have a child. At the same time, cloning could allow the selection of desirable characteristics (e.g. have better health by eliminating disease-causing genes).


Other than that, cloning could be used to eliminate the problems of immunological rejection during organ transplantation. This, therefore, removes the need for the patient to consume immunosuppressant drugs over their lifetime, which are needed to stop their body from attacking the transplanted tissue.


Arguments against human cloning

In general, the majority of the arguments against human cloning are linked to serious moral and ethical debates.


For instance, although cloning could be used as a form of infertility treatment that sounds somewhat harmless initially, there are huge concerns that this technology could be exploited for non-therapeutic reasons, in which genetically modified humans could be created for malicious purposes. Additionally, many people argued that cloning is unnatural and/or goes against “nature’s plans”.


Furthermore, cloning could restrict genetic diversity, and thereby reduce the frequency of advantageous alleles that aid survival (e.g. those that help to protect humans against pathogens). Plus, as showcased by Dolly’s biography, it was postulated that no artificial procedure can completely mimic a natural process and produce a consistent outcome. Thus, abnormal human phenotypes may arise and this may cause further health complications.


All in all, although the birth of Dolly the sheep presented exciting ideas on how cloning could be used to resolve health-related issues in humans, there still remains a myriad of questions regarding how cloning technologies ought to be utilised in the future to prevent any compromising on ethics.


Author

Ella Kline

BSc Biochemistry

Imperial College London


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