A Brief History of Cloning

27th April 2014

Colour photograph showing cells dividing.

Dividing cells showing chromosomes (purple) and cell skeleton (green). Image credit: Nasser Rusan/National Heart, Lung, and Blood Institute, National Institutes of Health/Public domain.

Earlier this month, scientists in South Korea and the United States announced that they had cloned a human embryo, which could have developed into a foetus if it had been implanted into a surrogate mother[1a].

Cloning is the process of producing genetically identical individuals. This happens naturally in all asexual reproduction and in sexual reproduction when identical siblings are born. Asexual reproduction is the primary method of reproduction for single celled organisms, and many plants and fungi. Insects like bees and ants can also reproduce asexually, as can some reptiles, fish, and birds[2].

When scientists refer to cloning, however, they are often referring to artificial cloning. This is most commonly achieved via a process known as somatic cell nuclear transfer (SCNT). Here you take the genetic code from one animal and place it into the egg cell of another. If the egg is then placed in a surrogate mother, it will develop into a foetus that's genetically identical to the animal the genetic code came from[3].

1. The genetic code

If you look at any of the different parts of an animal through a microscope, their bones, or organs, or muscles, for example, then you will find that they are all composed of tissue, which is created from cells. These cells can have different functions, but they all have a nucleus that contains the DNA of the animal they belong to[4a].

Single strands of DNA can wrap into a structure known as a chromosome, which we can see with electron microscopes. If you zoom in on a chromosome, you will see that a strand of DNA looks like it's actually made of two strands, held together by bars. These bars are composed of pairs of chemicals known as nucleobases.

There are four types of nucleobases known as adenine (A), thymine (T), cytosine (C), and guanine (G), and only A and T, and G and C, will pair with each other. This means that each half of a strand of DNA contains enough information to replicate itself. If one half has an A nucleobase, for example, then the other half must be T. DNA replicates within the nucleus of cells by splitting into two strands. These then make up the two halves of two new strands, and the rest of the structure is built around them.

Diagram of human with various types of tissue highlighted. Nervous tissue is found in the brain and spinal cord, muscle tissue in the heart and muscles, connective tissue in bones and tendons, and epithelial tissue in the skin and intestinal lining.

Image credit: OpenStax College/CC-A.

Diagram showing a chromosome within the nucleus of a cell. The chromosome can be unravelled to reveal a chain of DNA. This is shaped as a double helix composed of base pairs, designated T and A, and C and G.

Image credit: KES47/CC-A.

Almost all of an animal's physical characteristics are determined by proteins, which are made from chains of amino acids, and DNA contains information on which proteins to make. This information is encoded in the order of nucleobases, which are read in groups of three, along one-half of the DNA strand[4b].

The order G, C, A, for example, is code for the amino acid alanine, and A, G, A is code for the amino acid arginine, and so the code G, C, A, A, G, A means make a protein chain starting with the amino acid alanine followed by arginine.

Each set of three is known as a codon, and there are 64 possible codons, including codons stating where a chain begins and ends. The whole chain of codons, corresponding to a whole chain of amino acids needed to make a protein, is known as a gene.

A single strand of DNA forms a chromosome, and different species have a different number of chromosomes. Humans have 23 different types of chromosomes contained in the nucleus of their cells. Their somatic cells - that is almost any animal cell apart from their egg or sperm cells - contain two copies of each type of chromosome, one inherited from their biological mother, and one from their biological father. Their egg or sperm cells contain only one copy of each.

Humans are the same because our DNA is composed of genes that are arranged in the same order - in order of the tasks they perform - along the same number of chromosomes. We are different because we can have different genes perform the same tasks.

The gene HERC2 on the 15th human chromosome, for example, designates eye colour in humans, but humans have different eye colours, and so the code for this gene can differ from individual to individual. The difference in just one nucleobase, for example, results in the difference between a gene for blue eyes and a gene for brown eyes.

Each human has two copies of the 15th chromosome; one copy may have the gene for blue eyes and one copy may have the gene for brown eyes. The blue-eyed gene is said to be recessive because, if this is the case, then the person will have brown eyes. They will only have blue eyes if both chromosomes contain the blue-eyed gene.

After each strand of DNA has been replicated inside of a somatic cell, the cell can then divide, in a process known as mitosis[4c].

The production of egg and sperm cells are slightly different, and are formed in a process known as meiosis. Here, after the strands of DNA have replicated, sections of DNA on each chromosome, and its partner, are swapped. This process mixes traits inherited from the chromosomes provided by both of a person's biological parents. The cells then divide again, with chromosome pairs separating, so that each resulting egg or sperm cell only contains a single set of 23 chromosomes. The mixing of chromosomes means that the egg and sperm cells in each person are all different.

Diagram showing that DNA replicates by splitting in half and building new structure around each half.

DNA replication. Image credit: Madeleine Price Ball/CC-SA.

Diagram showing cells dividing during mitosis and meiosis. In the case of meiosis, gametes are produced.

Mitosis and meiosis. Image credit: John Schmidt/CC-SA.

When an egg is fertilised, it inherits one set of chromosomes from each biological parent, resulting in a somatic cell with 46 chromosomes[4d]. The resulting child will then have genes taken from all four of its biological grandparents. Identical twins are created if the fertilised egg then splits, if one half splits again, then identical triplets will be produced, and so on.

2. Cloning

2.1 Embryo twinning

The first cloning experiments produced clones by splitting the first two cells of a developing embryo, in a similar way to how identical twins are created naturally. German biologist Hans Driesch demonstrated that this could be achieved artificially for the first time in 1885, using sea urchins[5]. German embryologist Hans Spemann used this method to clone salamanders in 1902[6].

In 1928, Spemann showed that the genetic information needed for cloning is held in the nucleus of cells[7]. He did this by pushing the nucleus of a fertilised egg to side of the cell. He then used a strand of hair to separate the two sides, essentially splitting it into two cells. The cell with the nucleus then replicated itself while the other cell did not. When the hair was removed, the two cells merged again.

2.2 Somatic cell nuclear transfer (SCNT)

British biologist John Gurdon was the first to successfully clone an animal using SCNT in 1958[8a]. In SCNT, the nucleus of a somatic cell is taken from the animal you want to clone. It's then placed in the egg cell of another animal, which has had its nucleus removed. This egg then contains a nucleus with two sets of chromosomes, the same as a fertilised egg, and they are all identical to the animal you took them from.

Diagram showing the SCNT process, where a nucleus is removed from an egg cell, and replaced with the nucleus of a cell from the animal you wish to clone. In reproductive cloning, this is placed in a surrogate mother. In therapeutic cloning, the cells create a tissue culture.

Diagram of SCNT Process. Image credit: Wikibob/Schorschski/Dr Jürgen Groth/CC-SA.

This technique was later improved so that the nucleus was not physically removed from the somatic cell before being inserted into the egg. Instead, a small electric shock is used to fuse the two cells together. Danish embryologist Steen Willadsen developed this method in 1984, using embryotic rather than somatic cells[9a].

2.3 Therapeutic cloning

In therapeutic cloning, the egg then forms into a small mass of cells, an embryo, and in a few days embryotic stem cells develop. Embryonic stem cells have the ability to develop into any type of cell, and are naturally created in the fertilisation process in order to make different parts of the body. The embryo is prevented from developing further, and the stem cells can then be used for medical purposes[10].

A person could use SCNT to clone themselves in order to produce cells that are biologically compatible with their body, and so will not be rejected by their immune system. These cells can then be used to grow organs for organ transplantations, or used to treat any disease or illness that is caused by damage to specific cells. These include Alzheimer's disease, multiple sclerosis, Huntington's disease, and Parkinson's disease, which could all be treated with nerve cells, heart disease, which could be treated with heart cells, or blindness, which could be cured with photoreceptor cells.

Russian-American embryologist Alexander Maksimov first coined the term 'stem cells' in 1908 in order to describe the type of stem cells in the bone marrow of adults that can turn into different types of blood cells[11]. There was little further research until the 1960s, starting in 1961 when Canadian biologists James Till and Ernest McCulloch first demonstrated their existence[12]. Twenty years later, American biologist Gail Martin[13], and British biologists Martin Evans and Matthew Kaufman[14], separately and simultaneously discovered how to extract embryonic stem cells from mice.

American biologist James Thomson first extracted embryonic stem cells from humans in 1998[15]. Thomson used embryos left over from in vitro fertilization (IVF) procedures. In vitro fertilisation means fertilisation outside of the body (in vitro means 'in glass' in Latin). Physician John Rock and technician Miriam Menkin performed the first successful IVF of a human in 1944[16]. If the fertilised egg had been placed inside a surrogate mother, then they could have given birth to a 'test-tube baby'. The first test-tube baby, Louise Brown, was born in 1978[17].

2.4 Reproductive cloning

In reproductive cloning, the egg is placed in a surrogate mother and develops naturally. Eventually the surrogate mother will give birth to a child almost genetically identical to the person they wished to clone. SCNT does not produce completely genetically identical offspring because some of the DNA from the person or animal that donated the egg is left. This is because some DNA resides in mitochondria, which are located outside of the nucleus of the egg cell[18a].

Even if two animals were genetically identical, they may not be physically identical, or mentally similar, because of the large role the environment plays in shaping us. This can be seen in the differences between identical twins.

Reproductive cloning can be used for many things. Livestock with positive traits, such as lean meat, can be cloned to produce food, in a similar way to how we clone plants[19]. Endangered species can be kept alive through cloning, and extinct species can be brought back from the dead[18b].

There is also a market for cloned pets, and animals we find particularly physically appealing, like racehorses and pedigree dogs[20]. Cloned animals are also of use as scientific research subjects, since their responses to chemicals and drugs and should be uniform[21]. Clones are sometimes used in science fiction in order to provide their biological parent with organs, but it seems much more efficient to do this through therapeutic cloning.

The first animals to be cloned using nuclear transfer were frogs, which were cloned by American biologist Robert Briggs and Thomas King in 1952, taking the nucleus from an embryo[22]. British biologist John Gurdon became the first person to clone an animal using SCNT, in 1958[8b]. Gurdon used the same method as Briggs and King but took the nucleus from the somatic cell of a tadpole. Chinese embryologist Tong Dizhou cloned the first fish using SCNT in 1963[23].

The egg cells of mammals are much smaller than those of frogs or fish, and it took over 30 years before the first mammal was successfully cloned using SCNT. This was a sheep named Dolly, cloned by British biologists Ian Wilmut and Keith Campbell in 1996[24]. Willadsen had previously cloned a sheep in 1984, but he did not use somatic cells[9b]. In 1998, private company Advanced Cell Technology (ACT) claimed to have used SCNT to create a hybrid, using human somatic cells and the egg of a cow[25a]. The resulting embryo was reportedly destroyed after 12 days.

Many animals have been cloned using SCNT since the late 1990s, including cows[26], mice[27], pigs[28], and goats[29]. The first endangered species, the gaur, a type of ox, was cloned using SCNT in 2001, but it died just a few days after its birth[30]. The mouflon, a type wild sheep, became the first clone of an endangered species to survive beyond infancy, surviving for at least 7 months after its birth in 2001[31]. These were cloned using egg donors and surrogate mothers from similar, domesticated, species.

Diagram showing the SCNT process, where the nucleus is extracted from the somatic cell of a bucardo.

The process used to clone a bucardo in 2009. Image credit: 15ldavenport/CC-SA.

This technique allows extinct animals to be cloned if enough viable genetic material can be found. It might be possible, for example, to clone woolly mammoths if frozen cells are found, using elephants as egg donors and surrogates[32].

It's possible that genetic material can survive freezing. Another type of endangered ox, the Banteng, was successfully cloned in 2003 using the genetic material of a Banteng that had died 20 years earlier, and had been stored in San Diego Zoo's Frozen Zoo[33]. A frozen zoo stores the genetic material of endangered or extinct species in order to protect their genetic diversity.

The first extinct species was cloned in 2009[34]. This was a type of Spanish mountain goat called the bucardo, which went extinct in 2000. The bucardo was cloned using goats as egg donors and surrogates; however, it died soon after birth.

2.5 Genetic engineering

SCNT can be used to create genetically modified animals, as well as clones. In genetic modification, also known as genetic engineering, a gene is taken from one plant or animal, and is then physically placed within the DNA of another, which can then pass the gene on to its offspring. These two plants or animals do not have to be the same species. A clone created from the genetic material of more than one species is referred to as transgenic[35].

As well as having multiple medical and scientific uses[36], there's a market for genetically modified livestock[37], and other working animals, research subjects, and pets[38a].

Genetic engineering requires a more detailed knowledge of DNA than cloning, and was not possible until the 1970s.

DNA was not associated with genetic material until American geneticists Oswald Avery and Maclyn McCarty, and Canadian-American geneticist Colin MacLeod proved there was a connection in 1944[39]. American geneticists Alfred Hershey and Martha Chase confirmed DNA's role in reproduction in the early 1950s[40], and British chemist Rosalind Franklin[41] and American molecular biologist James Watson and British molecular biologist Francis Crick[42] showed that DNA has a double helix structure in 1953. American geneticist Joe Hin Tjio identified the number of chromosomes in humans in 1956[43].

Scientists did not have the ability to cut and paste sections of DNA until 1970, when American microbiologist Hamilton Smith and colleagues discovered enzymes that can do this naturally[44][45]. Belgian molecular biologist Walter Fiers and his colleagues were the first to determine the sequence of a gene, which belonged to a virus, in 1972[46]. Fiers determined the sequence of complete genome in 1976[47].

American geneticists Herbert Boyer and Stanley Cohen created transgenic bacteria in 1973[48], and German-American biologist Rudolf Jaenisch created the first transgenic mouse the following year[49].

Bacteria were genetically engineered in order to produce the protein insulin, which could then be used to treat people with diabetes, in 1978[50], becoming commercially available in 1982[51]. Plants such as tobacco, tomatoes, and potatoes were genetically engineered by the 1990s, and genetically modified plants are now commonly sold, and eaten, around the world[52]. In 1997, German biologist Angelika Schnieke, and the team behind Dolly the sheep, cloned a genetically modified sheep, Polly[53]. Polly was genetically modified to produce milk containing a protein essential for blood clotting in humans, which can be used to treat haemophilia.

In 1999, biologist Zhiyuan Gong and his colleagues at the National University of Singapore aimed to make fish that turned fluorescent when exposed to pollution[54]. They inserted the DNA of a tropical fish, known as the zebrafish, with the gene that makes certain types of jellyfish glow green under blue and ultraviolet light. This made the fish glow green in visible and ultraviolet light. They later produced a red fluorescent zebrafish using a gene from a sea coral[55].

The method was patented, and sold to Yorktown Technologies, L.P., a company in Texas, which branded them GloFish. GloFish became the first genetically modified pet to be sold, in 2003[38b]. Jellyfish genes have since been implanted into the DNA of mice[56], monkeys[57], pigs[58], and cats[59], in order to make them fluorescent.

The Human Genome Project, an international scientific research project formed with the goal of mapping all of the genes in the DNA of humans, was finally completed in 2003, after 13 years of research[60]. Since then, the genomes of many different life forms have been mapped, including those of fungi, plants, insects, crustaceans, fish, amphibians, reptiles, birds, and mammals[61]. The genomes of many endangered species have been sequenced, as have some extinct species, such as Neanderthals[62] and woolly mammoths[63].

2.6 Problems with SCNT

One problem with SCNT is that it has a low success rate when used for reproductive cloning. Hundreds of eggs were used in order to create Dolly the sheep, for example, and many cloned animals have died within a few days of their birth. We would not want to begin the reproductive cloning of humans until our success rate is markedly higher. Some people also object to therapeutic cloning using SCNT because it involves destroying an early stage embryo[64].

Both the therapeutic and reproductive cloning of humans using SCNT are problematic because they rely on egg donations from humans. Egg donation is a long and complicated procedure involving surgery, and so it's illegal to pay for egg donations for medical research in many parts of the world for the same reasons that it can be illegal to buy human organs. This can put female researchers in a vulnerable position, South Korean biologist Woo Suk Hwang, for example, was arrested in 2005, in-part for heavily pressuring the women that worked for him into donating eggs[65a].

While SCNT might work for the therapeutic cloning of individuals, there are simply not enough available eggs for the level of research required to cure diseases like Parkinson's disease.

The problems with therapeutic cloning using SCNT could all be resolved with another method for creating stem cells, which was discovered in 2006.

2.7 Induced pluripotent stem cells (iPSCs)

In 2006, Japanese biologists Kazutoshi Takahashi and Shinya Yamanaka discovered a new way to make stem cells that does not involve using donated eggs, or the creation and destruction of embryos[66]. Takahashi and Yamanaka discovered that if somatic cells are implanted with four specific genes, then they are reprogrammed back into embryonic-like stem cells. He named these induced pluripotent stem cells (iPSCs).

The term pluripotent refers to the fact that the stem cells have the potential to become any cell in the body; adult stem cells are not pluripotent and can only become a specific type of cell. The stem cells in our bone marrow, for example, can become any type of blood cell, but they cannot become anything else.

The term induced is used to differentiate Takahashi and Yamanaka's stem cells from embryonic stem cells, which are naturally pluripotent.

In 2007, Takahashi and Yamanaka announced that they had created iPSCs from the somatic cells of humans[67]. Thomson and his colleagues made the same discovery independently that year[68].

Diagram showing how induced pluripotent stem cells are created.

The technique for creating iPSCs, and embryonic stem cells using SCNT. Image credit: Kaidor/CC-SA.

One problem with iPSCs is that they can't be used to treat people with mitochondrial diseases. Mitochondria have their own DNA outside of the cell's nucleus. If a person with a mitochondrial disease wants to create stem cells then they can use SCNT to create embryonic stem cells, because in this procedure their genetic information is only taken from their cell's nucleus, mitochondrial DNA is taken from the donor egg.

If they created iPSCs instead, then their mitochondrial DNA would be reprogramed along with the DNA in their cell's nucleus, mutation and all.

In January 2014, Japanese biologist Haruko Obokata and colleagues claimed to have developed an easier way to reprogram cells into stem cells by exposing them to acid. They have since retracted their paper but are continuing to research this method[69].

3. Human Cloning

The first human-hybrid may have been cloned by Advanced Cell Technology in 1998 using the genetic material of a human and the egg of a cow; it was reportedly destroyed after 12 days[25b].

In 2005, Hwang claimed to have cloned the first human embryos, but his work was soon shown to be fraudulent and he was charged with embezzlement and violation of bioethics laws[65b].

American physician Samuel Wood and Australian biologist Andrew French cloned the first human embryo in 2008[70]. Woods cloned himself using a donated human egg, but did not succeed in creating stem cells. Biologists Dieter Egli and Scott Noggle became the first to produce human embryonic stem cells using SCNT, while working at the New York Stem Cell Foundation Laboratory, in 2011[71]. However, their process only worked when the eggs' nucleus remained in the cell. This meant that these embryos had three, rather than two, sets of chromosomes, and would not be compatible with their genetic donor.

American biologist Shoukhrat Mitalipov and colleagues created the first useful human embryonic stem cells using SCNT in 2013[72]. These produced stem cells for an 8-month-year-old with Leigh syndrome, a rare genetic disorder. Earlier this month, Young Gie Chung, Robert Lanza, and colleagues from the Research Institute for Stem Cell Research in Los Angeles, and the CHA Stem Cell Institute in South Korea repeated this process using adult somatic cells[1b].

While it generally seems more efficient to create stem cells using the iPSCs method, rather than via SCNT, this breakthrough will give researchers the opportunity to compare human stem cells created by the two methods.

These clones were created for therapeutic purposes, but the most recent could have developed into a foetus, had it been implanted into a surrogate mother. Human reproductive cloning is illegal in most countries around the world, and will probably remain so until the technique has a much better success rate.

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