National Institutes of Health
April 27, 1998
This paper presents background information on cloning. It includes a discussion of how cloning technologies are used today and how they could be used in the future. It also explains somatic cell nuclear transfer and describes the range of applications for this technology.
Cloning and somatic cell nuclear transfer are not synonymous. Cloning is the production of a precise genetic copy of DNA, a cell, or an individual plant or animal. Cloning can be successfully accomplished by using a number of different technologies. Somatic cell nuclear transfer is one specific technology that can be used for cloning. It is important to note that the use of somatic cell nuclear transfer is not limited to cloning an organism; there are other potential applications for both research and medicine that are discussed in this paper.
Definitions
- DNA—abbreviation for deoxyribonucleic acid which makes up genes.
- Gene—a functional unit of heredity which is a segment of DNA located in a specific site on a chromosome. A gene directs the formation of an enzyme or other protein.
- Cloning—the production of a precise genetic copy of a molecule (including DNA), cell, tissue, plant, animal.
- Somatic cell—cell of the body other than egg or sperm.
- Somatic cell nuclear transfer—the transfer of a cell nucleus from a somatic cell into an egg from which the nucleus has been removed.
Understanding the Biology of the Cell
Each human being has a unique set of genetic information that is inherited from his or her mother and father. All of this genetic information is found in the nucleus of the cell on structures called chromosomes. Each chromosome is made up of sequences or segments of DNA which are called genes. The absolute number of chromosomes differs between species—humans have 46. The entire genome, 46 chromosomes, is present in all cells of the human body, with the exception of sperm and egg cells, which each have 23 chromosomes. So, if every cell contains every gene, why do cells develop different function and appearances?
The explanation of how cells specialize begins with normal sexual reproduction and the development of an embryo. First, a sperm and an egg, each with 23 chromosomes, join together to form a fertilized egg with a full complement of 46 chromosomes—23 from the father and 23 from the mother. This fertilized egg has the potential to form any and every type of human cell; the fertilized egg is totipotent, or a cell with total potential. The fertilized egg cell divides and each new cell divides again. This process repeats itself many times.
After many such divisions, these totipotent cells begin to develop into a specific type of cell—a liver cell or a skin cell. The kind of cell is determined by what gene or genes are turned on in that particular cell. For example, if the genes that control liver formation are activated, the totipotent cell becomes a liver cell. But, if the genes that control muscle formation are turned on, that same totipotent cell can become a muscle cell. Under normal conditions, once the gene (or genes) that control muscle formation is activated, that cell loses the ability to become anything other than a muscle cell—it could never become a liver cell or a brain cell. In other words, the cell can never become totipotent again—at least that is what scientists once thought until the remarkable experiment that resulted in the birth of the sheep Dolly. But more about that later.
Cloning Today
DNA cloning
The simplest and most common form of cloning is DNA cloning, in which segments of DNA (usually a specific gene) are copied. A segment of DNA is inserted into a bacterium, and as the bacterium reproduces, identical copies of the DNA sequence are also produced. The multiple copies of DNA can be used for detailed studies of a specific gene. In addition, because genes direct the production of proteins, the bacteria with the inserted DNA can be triggered to act as miniature factories, churning out large quantities of a particular protein. For example, insulin is produced this way. The insulin gene is inserted into bacteria, and the bacteria become insulin factories, producing large amounts of insulin which are used for treatment. This is one type of recombinant DNA technology. Applications of DNA cloning in medical research and by the pharmaceutical industry are numerous, including the development of diagnostic methods for detecting diseases and disease-causing agents, the production of vaccines for preventing illness, and the development of antibiotics for fighting infections, among others. Specific examples of recombinant DNA produced products include: human insulin, human growth hormone, erythropoietin for kidney dialysis patients, clotting factor for hemophiliacs, and hepatitis B vaccine.
Tissue culture
Tissue culture is another common form of cloning. In tissue culture, a somatic cell (any cell of the body other than egg or sperm cells) is grown in the laboratory in culture dishes. As the cell divides, the newly replicated cells are clones, or identical copies, of the original cell. For years, cells from both animals and humans have been cultured in the laboratory. Cloned tissue culture cells have allowed scientists to test potential chemotherapies on cancerous cells, to study the cellular events leading to cancer, and to mass-produce drugs and vaccines. The cloning of cells in culture has reduced the use of live animals in research and has allowed studies of human cells that could not be done otherwise.
Growing cells in culture is, however, often difficult and is only successful with a limited number of specific types of cells. Some types of cells are difficult or even impossible to grow in culture. Other types of cells grow successfully in culture, but undergo major changes in their features or functions, i.e., they may no longer be representative of the tissue from which they were removed. Thus, tissue culture has limited usefulness. There is a great need for more effective means to develop human cell lines (cultures of specific types of cells) useful for both research and in the development of new ways to diagnose, treat, cure, or prevent a number of injuries and diseases.
Future Possibilities for Cloning
Significance of and therapeutic uses for somatic cell nuclear transfer
The experiment that created Dolly has the potential of expanding the applications of cloning technology to new areas of disease prevention, treatment and possibly cure. The February, 1997 report from Dr. Wilmut's laboratory introduced new concepts and theories regarding cellular differentiation. As described previously, once gene activation directs a totipotent cell to specialize into a specific type of cell, it had been thought that totipotency is lost; in other words, once "muscle genes" are activated, the cell will never produce anything other than muscle cells. The report of a technology called somatic cell nuclear transfer to create the sheep, Dolly, made scientists question this basic premise.
Somatic cell nuclear transfer as described by the Scottish scientists who created Dolly began when researchers took a normal sheep egg cell and removed the nucleus (cell structure containing the chromosomes). This left behind a sac of the fluid normally found inside an egg cell, which contains nutrients and other energy-producing materials that are essential for embryo development. Second, they selected a somatic cell, which is any cell other than an egg or a sperm cell—in this case, a cell from the mammary tissue of an adult sheep. Using carefully worked out laboratory conditions, the mammary cell was placed next to the egg from which the nucleus had been removed, and an electrical stimulus was applied to fuse the two cells.
Remarkably, when the resultant fused cell divided, rather than forming mammary cells, totipotent cells were formed, each with the full potential to become any kind of cell. These totipotent cells began to divide, and eventually, as is the case with a normal sexually-produced embryo, when implanted into the uterus of a ewe, the dividing, totipotent cells started to specialize, forming an embryo which developed into a fetus and ultimately a live-born lamb-a lamb that is genetically identical to the adult sheep from which the mammary cell was first taken! Although this may sound straightforward, in fact, it took 277 attempts to produce Dolly.
This experiment demonstrated that, when appropriately manipulated and placed in the correct environment, the genetic material of somatic cells can regain totipotency and when implanted into a uterus, form an embryo.
While news coverage of the Dolly experiment focused on the cloning of an animal from a single adult cell, biomedical scientists had a different focus. Medical scientists realized the genius of the Dolly experiment was the fact that the genome from a somatic cell was stimulated to return to a totipotent state. Scientists understood that somatic cell nuclear transfer could be used to investigate many of the complex events that occur during the development of normal cell differentiation, and possibly provide insight into the development of abnormal cells. For example, because some birth defects are caused by abnormal cell differentiation, if we knew why the process went wrong, perhaps we could learn how to prevent some of these birth defects. Improved understanding of cell differentiation could also provide knowledge on cell aging and regulation—these in turn may provide the clues needed for conquering debilitating and deadly diseases such as Alzheimer's disease and even cancer. In particular, researchers understood that if we knew how to turn on specific genes, the technology would permit the much-needed creation of cells for transplantation or grafting.
Diabetes
Consider a person with diabetes. These patients have a decreased number of insulin-producing cells in their pancreas. If we could augment these insulin-producing cells, we could potentially cure their diabetes. Cloning technology, using somatic cell nuclear transfer, might permit us to take a somatic cell from a diabetic patient and make that cell revert to its unspecialized state. If we then knew how to activate the genes which code for the production of an insulin-producing cell, we could create a large number of insulin-producing cells. These cells could then be transplanted back into the diabetic patient. Because the replacement cells would be genetically identical to the patient (cloned from the patient), there would be no problem with rejection.
This approach to the treatment of diabetes would be a vast improvement over the therapies available today. Rather than having to take insulin, pancreatic insulin cells could be replaced. In fact, a similar strategy is used today, but it has limited effectiveness. Insulin-producing cells are currently transplanted into patients with diabetes, but the only source of these replacement cells is through organ donation. Because these replacement cells are genetically different, the transplant patient must take powerful medications to prevent rejection. These medications to prevent rejection have severe and potentially fatal side effects and, often, rejection occurs despite the medication. Somatic cell nuclear transfer could revolutionize the treatment of this debilitating and often fatal illness.
Skin grafts
Somatic cell nuclear transfer might also be used in the future to create skin grafts for people who are severely burned. Patients with severe burns cannot survive without skin grafting. It is optimal to graft skin taken from unaffected areas of the same person's body. This is desirable, because it is genetically identical to the burn patient and, hence, will not be rejected. Unfortunately, when a patient has a large surface area burn, there is often not enough intact skin to use. Doctors can use skin from cadavers or skin cells grown in tissue culture. In both cases the skin is genetically different from the burn victim. While these provide material for emergency grafting, this skin is ultimately rejected and the patient must undergo grafting numerous times. Somatic cell nuclear transfer cloning could allow skin to be generated from virtually any of the burn victim's cells. The skin would be genetically identical and, hence, should not be rejected.
Both of these examples demonstrate the potential life-saving applications of somatic cell nuclear transfer—to produce vigorous healthy tissue for therapeutic purposes, not to clone a human being. It is important to note that, absent future research exploring this technology, these possibilities will be unrealized. Today, these are hopes for the future. In fact, there are many possible future applications of this technology. They include, for example:
- nerve stem cells to treat neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, Parkinson's disease, and to help repair injuries of the spinal cord
- bone marrow stem cells, for the treatment of leukemia or other blood diseases such as sickle cell disease
- liver cells to treat liver damage
- muscle cell precursors to treat muscular dystrophy and heart disease
- cartilage-forming cells to reconstruct joints damaged by injury or arthritis
Gene therapy
Somatic cell nuclear transfer could potentially be used as an adjunct to gene therapy. Gene therapy promises to be able to virtually replace a patient's defective gene with a normal gene, thereby curing or preventing disease. Currently, gene therapy researchers are developing ways to insert a copy of a normal gene into a virus that has been altered so that it should not cause disease. This modified virus is then introduced into the patient, where it will hopefully "infect" cells-without causing illness-and in the process "deliver" a normal gene into the cells of the patient. At present, the use of viruses as the courier for a normal gene is proving problematic. There are difficulties in transferring adequate amounts of the normal gene into patient cells and obstacles posed by the patient rejecting the courier virus.
Somatic cell nuclear transfer could potentially facilitate gene therapy by eliminating the need for a courier virus. A somatic cell from a diseased patient could be removed and a normal gene inserted using recombinant DNA techniques. The "corrected" cell could then be cloned using somatic cell nuclear transfer. The identical copies of the corrected cell could then be delivered back into the patient. Because the copies of the corrected cells are genetically identical to the patient, they should not be rejected. This technique would overcome some of the most difficult hurdles facing gene therapy today.
Animal Cloning
As Dolly showed the world, somatic cell nuclear transfer can also be used to clone animals. In traditional breeding practices, the offspring of an animal are sexually reproduced from genetically different parents and, therefore, may not share all of the characteristics that made the parents valuable. In conventional breeding, it takes years to produce many animals with similar genetic characteristics. Cloning could speed up this process and could allow the production of genetically identical animals.
This technique would be particularly valuable for research. The use of genetically identical animals could dramatically reduce the numbers of animals needed for experiments. For the first time, researchers could be certain that differences in responses to drugs and other interventions are due to the interventions, not to genetic differences between animals.
Cloning could also contribute to animal husbandry and medical research by facilitating transgenic technology. A transgenic animal is one that is genetically altered by inserting a new gene with the desired attributes into the DNA of a fertilized egg. Transgenic animals are valuable for a number of reasons. They can be engineered to have decreased susceptibility to bacterial infection, to have increased milk production, and to have the ability to produce pharmaceutically important proteins in their milk. Recently, calves were cloned with the gene that enabled them to produce milk containing human clotting factors which could be used for the treatment of hemophilia. Future advances may also allow the development of animal clones with tissues and organs that are compatible for human transplantation purposes. Cloning the animal that incorporated the gene of interest would be much faster than selective breeding and would decrease the amount of time required to produce transgenic animals.
*** In sum, somatic cell nuclear transfer holds many diverse and important possibilities to significantly improve prevention, treatment and perhaps the ability to cure disease. All of these possibilities can be accomplished without using this technology to create a human being.
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