Why are scientists interested in cloning?

The main reason that the scientists at Roslin wanted to be able to clone sheep and other large animals was connected with their research aimed at producing medicines in the milk of such animals. Researchers have managed to transfer human genes that produce useful proteins into sheep and cows, so that they can produce, for instance, the blood clotting agent factor IX to treat haemophilia or alpha-1-antitrypsin to treat cystic fibrosis and other lung conditions.

Cloned animals could also be developed that would produce human antibodies against infectious diseases and even cancers. ‘Foreign’ genes have been transplanted into zebra fish, which are widely used in laboratories, and embryos cloned from these fish express the foreign protein. If this technique can be applied to mammalian cells and the cells cultured to produce cloned animals, these could then breed conventionally to form flocks of genetically engineered animals all producing medicines in their milk.

There are other medical and scientific reasons for the interest in cloning. It is already being used alongside genetic techniques in the development of animal organs for transplant into humans (xenotransplantation). Combining such genetic techniques with cloning of pigs (achieved for the first time in March 2000) would lead to a reliable supply of suitable donor organs. The use of pig organs has been hampered by the presence of a sugar, alpha gal, on pig cells, but in 2002 scientists succeeded in knocking out the gene that makes it, and these ‘knockout’ pigs could be bred naturally. However, there are still worries about virus transmission.

The study of animal clones and cloned cells could lead to greater understanding of the development of the embryo and of ageing and age-related diseases. Cloned mice become obese, with related symptoms such as raised plasma insulin and leptin levels, though their offspring do not and are normal. Cloning could be used to create better animal models of diseases, which could in turn lead to further progress in understanding and treating those diseases. It could even enhance biodiversity by ensuring the continuation of rare breeds and endangered species.

What happened to Dolly?

Dolly, probably the most famous sheep in the world, lived a pampered existence at the Roslin Institute. She mated and produced normal offspring in the normal way, showing that such cloned animals can reproduce. Born on 5 July 1996, she was euthanased on 14 February 2003, aged six and a half. Sheep can live to age 11 or 12, but Dolly suffered from arthritis in a hind leg joint and from sheep pulmonary adenomatosis, a virus-induced lung tumour to which sheep raised indoors are prone. On 2 February 2003, Australia's first cloned sheep died unexpectedly at the age of two years and 10 months. The cause of death was unknown and the carcass was quickly cremated as it was decomposing.

Dolly’s chromosomes were are a little shorter than those of other sheep, but in most other ways she was the same as any other sheep of her chronological age. However, her early ageing may reflect that she was raised from the nucleus of a 6-year old sheep. Study of her cells also revealed that the very small amount of DNA outside the nucleus, in the mitochondria of the cells, is all inherited from the donor egg cell, not from the donor nucleus like the rest of her DNA. So she is not a completely identical copy. This finding could be important for sex-linked diseases such as haemophilia, and certain neuromuscular, brain and kidney conditions that are passed on through the mother's side of the family only.

Improving the technology

Scientists are working on ways to improve the technology. For example, when two genetically identical cloned mice embryos are combined, the aggregate embryo is more likely to survive to birth. Improvements in the culture medium may also help.

Ethical concerns and regulation

Most of the ethical concerns about cloning relate to the possibility that it might be used to clone humans. There would be enormous technical difficulties. As the technology stands at present, it would have to involve women willing to donate perhaps hundreds of eggs, surrogate pregnancies with high rates of miscarriage and stillbirth, and the possibility of premature ageing and high cancer rates for any children so produced. However, in 2004 South Korean scientists announced that they had cloned 30 human embryos, grown them in the laboratory until they were a hollow ball of cells, and produced a line of stem cells from them. Further news is awaited.

In the USA, President Clinton asked the National Bioethics Commission and Congress to examine the issues, and in the UK the House of Commons Science and Technology Committee, the Human Embryology and Fertilisation Authority and the Human Genetics Advisory Commission all consulted widely and advised that human cloning should be banned. The Council of Europe has banned human cloning: in fact most countries have banned the use of cloning to produce human babies (human reproductive cloning). However, there is one important medical aspect of cloning technology that could be applied to humans, which people may find less objectionable. This is therapeutic cloning (or cell nucleus replacement) for tissue engineering, in which tissues, rather than a baby, are created.

In therapeutic cloning, single cells would be taken from a person and 'reprogrammed' to create stem cells, which have the potential to develop into any type of cell in the body. When needed, the stem cells could be thawed and then induced to grow into particular types of cell such as heart, liver or brain cells that could be used in medical treatment. Reprogramming cells is likely to prove technically difficult.

Therapeutic cloning research is already being conducted in animals, and stem cells have been grown by this method and transplanted back into the original donor animal. In humans, this technique would revolutionise cell and tissue transplantation as a method of treating diseases. However, it is a very new science and has raised ethical concerns. In the UK a group headed by the Chief Medical Officer, Professor Liam Donaldson, has recommended that research on early human embryos should be allowed. The Human Fertilisation and Embryology Act was amended in 2001 to allow the use of embryos for stem cell research and consequently the HFEA has the responsibility for regulating all embryonic stem cell research in the UK. There is a potential supply of early embryos as patients undergoing in-vitro fertilisation usually produce a surplus of fertilised eggs.

Dolly, the cloned sheep

Dolly the sheep may have been the world's most famous clone, but she was not the first. Cloning creates a genetically identical copy of an animal or plant. Many animals - including frogs, mice, sheep, and cows - had been cloned before Dolly. Plants are often cloned - when you take a cutting, you are producing a clone. Human identical twins are also clones.

So Dolly was not the first clone, and she looked like any other sheep, so why did she cause so much excitement and concern? Because she was the first mammal to be cloned from an adult cell, rather than an embryo. This was a major scientific achievement, but also raised ethical concerns.

Since 1996, when Dolly was born, other sheep have been cloned from adult cells, as have mice, rabbits, horses and donkeys, pigs, goats and cattle. In 2004 a mouse was cloned using a nucleus from an olfactory neuron, showing that the donor nucleus can come from a tissue of the body that does not normally divide.

How was Dolly produced?

Producing an animal clone from an adult cell is obviously much more complex and difficult than growing a plant from a cutting. So when scientists working at the Roslin Institute in Scotland produced Dolly, the only lamb born from 277 attempts, it was a major news story around the world.

To produce Dolly, the scientists used the nucleus of an udder cell from a six-year-old Finn Dorset white sheep. The nucleus contains nearly all the cell's genes. They had to find a way to 'reprogram' the udder cells - to keep them alive but stop them growing – which they achieved by altering the growth medium (the ‘soup’ in which the cells were kept alive). Then they injected the cell into an unfertilised egg cell which had had its nucleus removed, and made the cells fuse by using electrical pulses. The unfertilised egg cell came from a Scottish Blackface ewe.

When the scientists had managed to fuse the nucleus from the adult white sheep cell with the egg cell from the black-faced sheep, they needed to make sure that the resulting cell would develop into an embryo. They cultured it for six or seven days to see if it divided and developed normally, before implanting it into a surrogate mother, another Scottish Blackface ewe. Dolly had a white face.

From 277 cell fusions, 29 early embryos developed and were implanted into 13 surrogate mothers. But only one pregnancy went to full term, and the 6.6kg Finn Dorset lamb 6LLS (alias Dolly) was born after 148 days.

Haemophilia and human inheritance

Take haemophilia for example:haemophilia is a disease where blood does not clot properly so people can in severe cases bleed to death. In the last century haemophilia was known as the Royal Disease because it was so common in the Royal Family. The reason for this was of selective breeding. The gene causing the problem is recessive and emerged when members of the European Royal Families continually inter-married.

The result was a "pedigree" with the same kind of problems as in over-bred animals. There is then a biological basis for the Biblical injunction against close relatives marrying. Cross-fert­ilisation is needed to keep us all healthy.

In those with haemophilia the substance which is missing is called Factor 8 - a substance which is found in normal blood and which is one component of the clotting mechanism.Factor 8 can be extracted from blood donated for blood transfusion, although the process is complicated and expensive. If someone with haemophilia is bleeding uncontrollably from a cut an injection of Factor 8 stops it very well.

The Extraction process has turned out to be very unsafe however: all over the world, the virus causing AIDS (called HIV) found it's way into donated blood. Whereas an infected blood transfu­sion to an uninfected person only results in one new infection, Factor 8 is obtained by pooling plasma from a very large number of people.

Just one donation in a hundred can be enough to contaminate the whole process so that dozens become infected from the injectedFactor 8. One reason why Factor 8 supplies before 1985 were so dangerous is that the UK depended on Factor 8 imported from the US. In that country blood donors are paid with the result that many drug addicts donate blood to raise extra income. This greatly increased the risk of hepatitis B virus or HIV finding their way in to the blood banks. Effective testing from 1985 (using techniques derived from Genetic engineering) has almost eliminated this risk. However, by 1985 in the UK over 100 men and 250 boys were already HIV infected through these treatments.

In addition to blood testing, special treatments since 1985 have made Factor 8 particularly safe. Nevertheless the pressure has been growing to make Factor 8 in the laboratory. In 1984 the genes programming for Favtor 8 were identified for the first time, copied, and analysed (10). In the last two to three years Genetic engineering has now been used to programmecells from mammals grown in the laboratory or in the factory to produce human factor 8 (20). We will be looking at this remarkable achievement in more detail later on.

Why are all those with haemophilia men? Mendel's experiments explain to us why:most people have a pair of genes to tell cells how to make Factor 8.Even if one gene is faulty or missing, the process can continue.If, however, as a result of an unlikely and unhappy accident, a man and woman who both have a faulty gene have a family then Mendel would tell us that on average one in four of their children will inherit two faulty genes and be unable to produce Factor 8.Two others will be carriers and the fourth will have both genes intact.

The interesting thing about haemophilia is that the gene carrying information on blood clotting just happens to be sitting on the X or female chromosome.This "linkage" of one characteristic (sex) with another (clotting) is extremely important to the geneticengineer as we shall see later on.Linkage with an outward obvious sign is a good marker of other genes also inherited in the "package".

THE GENETIC REVOLUTION

From the most ancient times a rule of life has been seen to be true: insects breed insects, birds breed birds, cows breed cows and humans breed humans. If you take acorns from an oak tree and plant them the result is more oak trees. Creatures and plants remain true to type, faithfully passing on their characteristics from generation to generation. Where thereare slight variations, for example in skin pigmentation or in the colouring of flowers, then these too mcan usually be traced down the generations. The basis of life has been remarkably stable considering its complexity.

The basis of this inheritance was not understood however. An understanding of how organisms are built out of cells only emerged with the invention of the first light microscope by Robert Boyle in the eighteenth century (check date). It was many decades later before we began to understand how the cell works. Most of the structures in a cell could only be seen with the high power of the electron microscope.

However, for many centuries experiments were already taking place with cross-breeding - the earliest technique of genetic engineer­ing.

In order to understand the mechanism of inheritance, we need to start in an Austrian monastery around 1760, in the potting sheds of a gardener called George Mendel.This monk was curious to know what would happen if he took pollen from one type of plant and used it to fertilise another.Would the pollen be accepted?Would it succeed in fertilising the plant?If it did, would seed result which would germinate?Finally, when it germinated, what kind of plant would grow?

For thousands of years previously such attempts had been made with animals.For instance, in the time of Jesus, it was common to allow a horse to mate with a donkey:the result was a cross-fertilised egg which went on to develop into a rather strange-looking creature at birth.The creature had some of the best characteristics of both parents and was known as a mule. This new species had one important drawback: you could not breed from it because it was always sterile.

Hundreds of others examples could be given over previous centuries of selective breeding - indeed Jacob in the Old Testament seemed to know what he was doing in selectively breeding white and black sheep to produce a herd entirely coloured as he wanted, at a time when sheep ownership was being determinedsolely by colouring of their woolen coats.

The process of inheritance has been well understood by families who observe - say -grandpa's orange hair through to a grand­child or other family likenesses.However, the mechanism has only relatively recently been fully understood.Why was it that dark haired parents could occasionally produce a fair-haired child ?

Mendel was interested in all this. Moreoverd the monastery stood to gain from improved strains of cereal plants. Mendel found that when he cross-fertilised closely related plants with obvious differences, he got neither a mix nor equal numbers of aech type. Instead he found a curious pattern. After a while he found he could predict in advance not only what variations he would see, but also how many of them.He realised that in each seed there was a lot more information stored than would ever be used to form the new plant.

Some of this information it seemed was hidden away in many plants and only expressed when cross-fertilisation took place. It seemed like each plant had its own strong and weak features. Weak features only came to the surface under certain circumstances. These strong features have become known as "dominant" while those which tend to be hidden away are called "recessive".

This same information and understanding is used daily in dozens of Genetic engineering laboratories all over the world every day. When he cross-ferti­lised tall and short varieties of the same plants he found he always landed up with seeds that produced plants in a fixed ratio of three tall to one short (check which way round). From this he prposed a theory which was to revolutionise our thinking about breeding.

He came to the conclusion that each plant must have two sets of instruc­tions for each part of its structure. Therefore each plant had two set of instructions for height. However if the plant had a mixture, then the tall one was always dominant.

You can see how this works in Fig 1. When sperms or eggs are made - or their equivalent in plants - the original cells divide into two, with only half the full set of instructions needed for life in each half. So parents with a mixture of tall and short instructions in their cells will produce sperm or eggs with either one or the other.

Fertilisation happens when pollen and ova meet, (or sperm and eggs in animals). When this happens, the new composite cell ahs a complete set of instructions and is able to start forming a new plant. Clearly four types of plants could result: one type where both pollen and ova have provided tall instructions, another where both are short, and two where there is a mix. Three of these out of four will be taall. The only plant type that will turn out short will be the one where both sets of instructions are short, because both parent plants passed on the recessive gene.

Fig 1

"Mother" "Father"

T S T S

Both these plants have a tall gene in the pair so both are tall.

Fig 2

1. Mother Father

T T A tall plant

2. Mother Father

T S A tall plant

3. Mother Father

S T A tall plant

4. Mother Father

S S A short plant

So in his classic experiment:two tall plants cross-fertilised produced short plants one time in four.Interestingly,if short plants are only fertilised by other short plants then you can see that no more tall plants will ever be produced.A new strain will have been created.

Simple methods like this have been widely used by gardeners and horticul­turists for over a hundred years: selective breeding from plants showing the characteristics you want to encourage. The development of pedigree dogs is an ancient art which has worked on the same principle: only allowing dogs to mate that have the right characteristics.

Incidentally you can see straight away a major problem: if you go on inter-breeding from just one small group, then more and more recessive genes may emerge. Some may have hidden dangers for the animal. Take dogs again as an example: in the wild they breed widely producing a group of fairly even appearance. If bad traits emerge , they tend to be eliminated because they dogs do not survive long enough to breed or because the recessive traits are covered up by dominant genes from others in the group. However in domestic breeding, the dominant genes are being deliberately trimmed out. The result is a beautiful breed but one which may be susceptible to a high rate of blindness, tumours or hip problems for example. Ther are many inherited disorders in humans that can arise in a similar way.

Human cloning

Human cloning is the creation of a genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. A third type of cloning called replacement cloning exists in theory, and is a combination of therapeutic and reproductive cloning. Replacement cloning entails the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant. It has been proposed as a way to greatly extend lifespan.

Human cloning is among the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning field to be halted. Some people and groups oppose therapeutic cloning but many more oppose reproductive cloning. The American Association for the Advancement of Science (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved . Serious ethical issues have arisen in discussions of harvesting of organs from clones. Some people have considered the idea of growing organs separately from a human organism - in doing this, a new organ supply could be established without the moral implications of harvesting them from human organisms. Research is also being done on the idea of growing organs that are biologically acceptable to the human body inside of other organisms, such as pigs or cows, then transplanting them to humans.

Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells which have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonally distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

Molecular cloning

Cloning is the process of creating an identical copy of something. In biology, it collectively refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also encompasses situations whereby organisms reproduce asexually.

Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is utilised in a wide array of biological experiments and practical applications such as large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence.

In essence, in order to amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, a sequence element capable of directing the propagation of itself and any linked sequence. In practice, however, a number of other features are desired and a variety of specialised cloning vectors exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

Cloning of any DNA fragment essentially involves four steps: fragmentation, ligation, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected, these are summarised as a ‘cloning strategy’.

Initially, the DNA of interest needs to be isolated to provide a relevant DNA segment of suitable size. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (α-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.