Cloning Genes

Gene cloning, or molecular cloning, has several different meanings to a molecular biologist. A clone is an exact copy, or replica, of something. In the literal sense, cloning a gene means to make many exact copies of a segment of a DNA molecule that encodes a gene. This is in marked contrast to cloning an entire organism—regenerating a genetically identical copy of the organism—which is technically much more difficult (with animals) and can involve ethical ramifications not associated with gene cloning. Molecular biologists exploit the replicative ability of cultured cells to clone genes.

Purposes of Gene Cloning

To study genes in the laboratory, it is necessary to have many copies on hand to use as samples for different experiments. Such experiments include Southern or Northern blots, in which genes labeled with radioactive or fluorescent chemicals are used as probes for detecting specific genes that may be present in complex mixtures of DNA.

Cloned genes also make it easier to study the proteins they encode. Because the genetic code of bacteria is identical to that of eukaryotes , a cloned animal or plant gene that has been introduced into a bacterium can often direct the bacterium to produce its protein product, which can then be purified and used for biochemical experimentation. Cloned genes can also be used for DNA sequencing, which is the determination of the precise order of all the base pairs in the gene. All of these applications require many copies of the DNA molecule that is being studied.

Gene cloning also enables scientists to manipulate and study genes in isolation from the organism they came from. This allows researchers to conduct many experiments that would be impossible without cloned genes. For research on humans, this is clearly a major advantage, as direct experimentation on humans has many technical, financial, and ethical limitations.

Cloning Techniques

Cloning genes is now a technically straightforward process. Usually, cloning uses recombinant DNA techniques, which were developed in the early 1970s by Paul Berg, of Stanford University, and, independently, by Stanley Cohen and Herbert Boyer, of Stanford and the University of California. These researchers devised methods for excising genes from DNA at precise positions, using restriction enzymes and then using the enzyme known as DNA ligase to splice the resulting gene-containing fragment into a plasmid vector .

Plasmids are small, circular DNA molecules that occur naturally in many species of bacteria. The plasmids naturally replicate and are passed on to future generations of bacterial cells. To replicate, all plasmids must contain a sequence, called an origin of replication, which directs the bacterial DNA polymerase to replicate the DNA molecule. In addition, recombinant plasmids contain one or more selectable markers. A selectable marker is a gene that confers on the bacterium harboring the plasmid the ability to survive under conditions in which bacteria lacking the plasmid would otherwise die. Usually, such genes encode enzymes that enable the bacterium to live and grow despite the presence of an antibiotic drug.

The recombinant plasmid is then introduced into a host cell, such as an Escherichia coli bacterium, by a process called transformation, and the cell is allowed to multiply and form a large population of cells. Each of these cells harbors many identical copies of the recombinant plasmid. The cells are then cultured in growth media containing the antibiotic to which the plasmid confers resistance. This ensures that only cells containing the recombinant plasmid will survive and replicate. A researcher then harvests the cells and can extract and purify many copies of the plasmid.

Another method to produce many copies of a DNA molecule, which is even simpler than traditional recombinant cloning methods, is the polymerase chain reaction (PCR). PCR amplifies the DNA in a reaction tube without the need for a plasmid to be grown in bacteria.

Importance for Medicine and Industry

The ability to clone a gene is not only valuable for conducting biological research. Many important pharmaceutical drugs and industrial enzymes are produced from cloned genes. For example, insulin, clotting factors, human growth hormone, cytokines (cell growth stimulants), and several anticancer drugs in use are produced from cloned genes.

Before the advent of gene cloning, these proteins had to be purified from their natural tissue sources, a difficult, expensive, and inefficient process. Using recombinant methods, biomedical companies can prepare these important proteins more easily and inexpensively than they previously could. In addition, in many cases the product that is produced is more effective and more highly purified. For example, before the hormone insulin, which many diabetes patients must inject, became available as a recombinant human protein, it was purified from pig and cow pancreases. However, pig and cow insulin has a slightly different amino acid sequence than the human hormone. This sometimes led to immune reactions in patients. The recombinant human version of the hormone is identical to the natural human version, so it causes no immune reaction.

Gene cloning is also used to produce many of the molecular tools used to study genes. Even restriction enzymes, DNA ligase, DNA polymerases, and many of the other enzymes used for recombinant DNA methods are themselves, in most cases, produced from cloned genes, as are enzymes used in many other industrial processes.

Genomic Versus cDNA Clones

A gene can take varying forms, and so can gene clones. The proteincoding regions of most eukaryotic genes are interrupted by noncoding sequences called introns, which are ultimately excluded from the mature messenger RNA (mRNA) after the gene is transcribed. In addition to the protein-coding sequences, all genes contain "upstream" and "downstream" regulatory sequences that control when, in which tissues, and under what circumstances the gene is transcribed. A clone containing the entire region of a gene as it exists on the chromosome, including introns and nontranscribed regulatory sequences, is called a genomic clone because it is derived directly from genomic, or chromosomal, DNA.

It is also possible to clone a gene directly from its messenger RNA transcript, from which all introns have been removed. This type of clone, called a complementary DNA or cDNA clone, includes only the protein-coding sequences and upstream and downstream sequences that do not code for amino acids but that may control how the mRNA transcript gets translated to protein.

To prepare cDNA a researcher starts with mRNA and then makes a complementary single-stranded DNA copy using the enzyme reverse transcriptase. Reverse transcriptase is a DNA polymerase that synthesizes DNA based on an RNA template that is produced by retroviruses. After the mRNA strand is digested away by another enzyme, called RNase H, DNA polymerase can synthesize a second DNA strand by using the newly made first strand cDNA as a template.

Because cDNAs lack introns, the protein-coding region in a cDNA molecule is contained in a single uninterrupted sequence, called an open reading frame, or ORF. This makes cDNA clones extremely useful for predicting the amino acid sequence of the protein that a gene encodes. It also makes it possible to direct protein synthesis from a eukaryotic cDNA clone in a bacterium, which cannot splice introns. With introns still present in a cloned gene, the bacteria will misinterpret the intron sequences as protein-encoding sequences. The resulting incorrect messanger RNA will encode a protein with an incorrect amino acid.

"Gene Cloning" Usually Means "Gene Identification"

When researchers report in a scientific journal that they have "cloned a gene" they are not referring to the rather mundane process of amplifying copies of a DNA molecule. What they are really talking about is the molecular identification of a previously unknown gene, and determination of its precise position on a chromosome. There are many different methods that can be used to identify a gene. Two of the most common approaches are discussed below.

A gene can be defined in several ways. In fact, the concept of the gene is undergoing a re-evaluation as scientists are analyzing the complete genomes of more and more organisms and finding that many sequences encode more than one protein product. Gregor Mendel identified genes—for example, he identified the factor that made peas either yellow or green—long before he or anyone else knew that genes were encoded on segments of the DNA that made up chromosomes. Studying genetics in the fruit fly, Drosophila melanogaster, Morgan and Sturtevant demonstrated that genes are entities that reside at measurable locations, or loci, on chromosomes, although they did not yet understand the biochemical nature of genes.

Modern geneticists often use the same methods as Mendel and Morgan to identify genes by physical traits, or phenotypes, that mutations in them can cause in an organism. But today we can go even further. Using a broad range of molecular biology techniques, including gene cloning, researchers can now determine the precise DNA coding sequence that corresponds to a particular phenotype . This capability is tremendously powerful, because discovering the gene responsible for a trait can help humankind understand the cellular and biochemical processes underlying the trait. For example, geneticists have learned a great deal about the basis of cancer by identifying genes that, when mutated, contribute to cancer. By studying these genes, researchers now know that many of them control when cells divide (e.g., proto-oncogenes and tumor suppressor genes) or when they die (e.g., the apoptosis genes). Under some circumstances, when such genes are damaged by mutation, cells divide when they shouldn't, or don't die when they should, leading to cancer.

Positional Cloning

Positional cloning starts with the classical methods developed at the turn of the twentieth century by Thomas Hunt Morgan, Alfred Sturtevant, and their colleagues, of genetically mapping a particular phenotype to a region of a chromosome. A detailed discussion of genetic mapping is beyond the scope of this section, but, in general, it is based on conducting genetic crosses between individuals with two different mutant traits and analyzing how often the traits occur together in the progeny of subsequent generations.

Genetic mapping provides a general idea of where a gene is located on a particular chromosome, but it does not identify the precise DNA sequence that encodes the gene. The next step is to locate the gene on what is called the physical map of the chromosome. A physical map is a high-resolution map of all the DNA sequences that make up a chromosome. One type of physical map is a restriction map, which depicts the order of DNA fragments produced when a large DNA molecule is cut with restriction endonucleases (restriction enzymes).

Restriction maps have been made for the complete genomes of several model genetic organisms, such as the fruit fly (Drosophila melanogaster ), and the roundworm, (Caenorhabditis elegans ). For these organisms, individual large DNA fragments—on the order of forty to one hundred thousand base pairs from the whole genome—have been cloned in bacterial plasmid vectors to make a "library" of the genome. Each fragment is mapped to a known position, but the identify of the gene or genes it contains is originally unknown. To identify the genes, a cloned fragment is introduced into a mutant fly or roundworm.

To pinpoint the location of a particular gene, a researcher can introduce one or several of the plasmid clones from the physical map that are in the general vicinity of the region on the genetic map where the gene is thought to lie into a mutant that is defective in the gene of interest. If the introduced DNA corrects the mutant's defect, that DNA probably contains a normal copy of the defective gene. But these large clones usually contain several genes. By further "trimming" the DNA into smaller subfragments and testing the ability of each subfragment to rescue mutants, the researcher can eventually home in on the gene. As further confirmation that this gene is the cause of the mutant phenotype, the researcher can isolate the corresponding gene from the mutant and determine its DNA sequence to see if it contains a mutation (a DNA sequence alteration) relative to the normal gene sequence.

Expression Cloning

In some cases, a researcher becomes interested in studying a gene not because mutations in it cause an interesting phenotype but because the protein it encodes has interesting properties. A prominent example is beta-amyloid protein, which accumulates in the brains of Alzheimer's disease patients.

Expression cloning is a method of isolating a gene by looking for the protein it encodes. If the protein of interest is an enzyme, it can be found by testing for its biochemical activity. A very common method for identifying a particular protein is by using antibodies, or immunoglobulins, that bind specifically to that protein. Expression cloning usually uses a cDNA library, in which protein-coding sequences are uninterrupted by introns. Each cDNA is inserted into an "expression vector," which contains all the necessary signals for the DNA to be transcribed into mRNA. The mRNA can then be translated into protein. Thus the host cell harboring the clone will produce the gene's protein product, and the protein can then be detected by biochemical or immunologic methods. Once the cell making the protein is found, the cDNA can be re-isolated and the gene sequenced by standard means.

Gene cloning techniques continue to advance rapidly, aided by the Human Genome Project and bioinformatics. It is likely that positional cloning will take on a secondary role, and that bioinformatics and proteomics methods will begin to contribute more, as more progress in these fields is made.

see also Bioinformatics; Blotting; Chromosomes, Artificial; Cloning Organisms; Cloning: Ethical Issues; DNA Libraries; Gene; Gene Discovery; Human Genome Project; Linkage and Recombination; Marker Systems; Morgan, Thomas Hunt; Plasmid; Polymerase Chain Reaction; Recombinant DNA; Restriction Enzymes; Reverse Transcriptase; RNA Processing; Sequencing DNA; Transformation.

Paul J. Muhlrad

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman and Company, 2000.

Micklos, David A., and Greg A. Freyer. DNA Science: A First Course in Recombinant DNA Technology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1990.

Watson, James D., et al. Recombinant DNA, 2nd ed. New York: Scientific American Books, 1992.

Cloning Organisms

There are two distinct types of cloning: molecular and organismal. Molecular cloning is the removal of a stretch of DNA, usually a gene, from an organism, and its insertion into another piece of DNA, such as a plasmid , to form a substance called recombinant DNA. This recombinant DNA may then be expressed in, or simply carried passively by, another organism, such as bacteria. Organismal cloning, the subject of this entry, is the production of genetically identical organisms and, as such, can be used to produce genetically identical copies of livestock or may be used to produce new members of endangered or even extinct species. It may be especially cost-effective to clone animals that produce therapeutic proteins such as blood clotting factors, thus combining both types of cloning. Cloning is controversial, however, because our understanding of the procedures needed to clone mammals may be applied to human cloning, which gives rise to profound ethical issues.

The History of Cloning

Cloning has a long history. Animals that reproduce sexually produce clones whenever identical twins are born. These twins are genetically indistinguishable, and are formed when a fertilized egg separates at a very early stage of development. Clones are also the natural product of asexual reproduction, although in this case perfect clones cannot be maintained through an infinite number of generations, because spontaneous mutations can and do occur. Lastly, clones can be produced by regeneration in both plants and animals. For example, plant cuttings will regenerate roots and, ultimately, an entire "new" plant, and some invertebrates, such as planaria, can regenerate two identical animals if the adult is cut in half. In these forms, cloning has been with us for a very long time.

Since the mid-1960s, scientists have been able to culture plant cells, that is, grow cells from plants such as tobacco and carrots in a petri dish, to get thousands of genetically identical cells. From such cultured cells an unlimited quantity of cloned plants can then be grown. These cultured cells can be modified to contain recombinant, or cloned, DNA as well.

Cloning Amphibians

The first cloning of a vertebrate by nuclear transfer was reported by John Gurdon of the University of Cambridge in the 1950s. In nuclear transplantation, the nucleus of an unfertilized donor egg is either mechanically removed or it is destroyed by ultraviolet light in a process called enucleation. The original nucleus is then replaced by a nucleus containing a full set of genes that has been taken from a body cell of an organism. This procedure eliminates the need for the fertilization of an egg by a sperm.

The most successful nuclear transplants have been achieved after serially transferring donor intestinal nuclei, that is, putting an adult nucleus from an intestinal cell into an egg whose nucleus was destroyed, allowing the egg to divide only a certain number of times, removing nuclei from these cells, and repeating this process several times before allowing the embryo to complete development. Eventually, transplantation of nuclei from albino embryonic frog cells into enucleated eggs from a dark green female frog led to the production of adult albino frog clones, demonstrating that a properly treated adult nucleus could support the full development of an egg into an adult clone. Later experiments demonstrated that nuclei from cells of other tissues, even quiescent cells such as blood cells, could also be used if properly treated. Despite these successes, no adult frog has been cloned when a nucleus from an adult cell was used without serial transfer. Without serial transfer of the nuclei, the animals would only develop to the tadpole stage, and then they would die.

Cloning of Mammals: Dolly

Nuclear transplantation has also been successful in producing mammalian clones, most notably of sheep, cattle, pigs, and mice. The most famous cloned mammal is a sheep named "Dolly," the first animal to be cloned directly from an adult cell. Experiments leading to the birth of Dolly were done at the Roslin Institute with collaborators at Pharmaceutical Proteins Limited, both in Scotland. This group had earlier produced Megan and Morag, the first mammals to be cloned from cultured cells. These two sheep were produced from embryonic cells, however, not from cells of an adult animal.

Dolly was born in the summer of 1996, the product of a nucleus from the mammary gland of a six-year-old female Finn-Dorsett sheep and an egg from a Scottish Blackface female. Mammary gland cells were grown in a petri dish and were deprived of nutrients so that they would stop dividing, just like an unfertilized egg. Donor eggs were taken from sheep soon after ovulation , and nuclei were mechanically removed from them. These enucleated eggs were then fused with the cultured mammary gland cells so that a mammary gland nucleus would be inside an unfertilized egg. Two hundred and seventy-seven such embryos were constructed and temporarily allowed to divide in a petri dish, and then all of them were transferred into the oviduct of a temporary surrogate mother. Of the original 247 embryos, only 29 developed further, and these were transferred to 13 hormonally treated surrogate mothers.

Only one surrogate mother became pregnant, and she only had one live lamb, named Dolly. The success rate was very low, but Dolly has been proven to be a true clone: She has all the characteristics of a Finn-Dorsett sheep. Independent scientists used a technique called DNA fingerprinting to show that Dolly's DNA matched the donor mammary cells but did not match that of other sheep in the Finn-Dorsett flock, nor did her DNA match that of her surrogate mother or the egg donor. Similar results have been obtained by Ryuzo Yanagimachi at the University of Hawaii, who worked with several generations of cloned mice.

In 1997 Polly, a sheep created with a combination of both molecular and organismal cloning techniques, was born. Polly was derived from a fetal sheep cell that had been engineered to contain the human gene that makes coagulation factor IX. Factor IX is missing in people with a disease called hemophilia type B. Polly and two other sheep were engineered to produce factor IX in their milk, thus providing people with hemophilia access to a safer and less expensive source of clotting factor than was previously available. Because Polly was made from more easily cultured and, therefore, more easily engineered embryonic cells, it is thought that this type of cloning technology holds the most promise for the future of pharmaceutical production of proteins that cannot be made in bacteria.

In January 2001, the first cloned member of an endangered species was born. This was a gaur, a wild ox native to India and southeast Asia, which the researchers named Noah. The gaur was chosen by Advanced Cell Technology as a candidate for cloning after the company had successfully cloned domestic cattle, which are related to the gaur species.

The embryo from which Noah developed was created from the nuclei of frozen skin cells that had been taken from an adult male gaur that had died eight years earlier. Skin cell nuclei were fused with enucleated domestic cow eggs to produce forty embryos. One of these forty was carried to full term in a surrogate cow mother. Unfortunately, Noah died of an infection two days after his birth (the infection is thought to be unrelated to his origin as a cloned animal). Despite Noah's death, it is likely that cloning will eventually be used to aid the conservation of endangered species. In the future, scientists may attempt to clone a recently extinct species, should intact DNA for an extinct species be obtained.

Problems with Cloning

In general, the success rate of mammalian cloning is low, with less than 0.1 to 2.0 percent of transplanted nuclei yielding a live birth. The vast majority of transplants fail to divide or to develop normally, indicating there is much we still do not understand about reprogramming an adult nucleus to support embryonic development. One thing that is clear, however, is that having both the donor cell and host egg cell in a nondividing state is essential for success.

What might be both the most vexing and most interesting problem with cloning is related to aging. Chromosomes "show their age" by a shortening in their tips, or telomeres , a process that occurs every time the cell they are in divides. This telomere shortening occurs in all cells except eggs, sperm, and most cancer cells, and shortened telomeres are correlated with the aging of organisms. Since the nuclear DNA in most cloned animals is taken from an adult, the chromosomes of cloned animals are expected to have shorter telomeres than animals of the same birth age that are produced by sexual reproduction, causing researchers to wonder whether cloned animals will age prematurely. Shorter telomeres have been found in Dolly and other cloned sheep, but telomeres are reported not to be shorter in cloned mice or cattle. Underlying reasons for the different results may include differences between cell types or species used.

The Myth of the Perfect Clone

Cloned animals are not 100 percent identical to their "parents." Whenever nuclear transplantation is used to produce cloned organisms, the offspring display some differences from the organism that donated the nuclei. The egg donor contributes mitochondria, the energy producers of eukaryotic cells, and these mitochondria have their own small amount of DNA-containing genes used for energy metabolism. Since mitochondria are inherited only with egg cytoplasm, they will not match the mitochondria of the animal from which the nucleus was taken. In addition, maternally derived gene products, both mRNA (messenger RNA) and protein, which serve to begin embryonic development, will differ from that of the nuclear donor, as will the uterine environment and the external environment. Thus, for example, clones produced by nuclear transplantation will be significantly less identical than will clones produced by twinning.

see also Cloning: Ethical Issues; Cloning Genes; Conservation Biology: Genetic Approaches; Hemophilia; Mitochondrial Genome; Reproductive Technology; Telomere; Transgenic Animals; Twins.

Elizabeth A. De Stasio

Bibliography

Gurdon, J. B., and Alan Colman. "The Future of Cloning." Nature 402 (1999): 743.

Lanza, Robert P., Betsy L. Dresser, and Philip Damiani. "Cloning Noah's Ark." Scientific American (Nov., 2000): 84-89.

Wilmut, Ian. "Cloning for Medicine." Scientific American (Dec., 1998): 58-63.

Wilmut, Ian, Keith Campbell, and Colin Tudge. The Second Creation: Dolly and the Age of Biological Control. Cambridge, MA: Harvard University Press, 2000.

Cloning

Cloning burst upon the scene in February, 1997, with the announcement of the birth of Dolly, the cloned sheep. She was created when researchers took the DNA nucleus from a cell of an adult sheep and fused it with an egg from another sheep. Shortly after Dolly was born, mice, cattle, goats, pigs, and cats were also cloned.

For biologists, however, the word cloning refers not to producing new animals but rather to copying DNA, including short segments such as genes or parts of genes. This ability to copy DNA is a basic technique of genetic engineering used in almost every form of research and biotechnology. In Dolly, copying was taken to the ultimate scale, the copying of the entire nucleus or the entire genome of the sheep. The transfer of the nucleus is usually called somatic cell nuclear transfer (SCNT), and this is what most people have in mind when they speak of cloning.

Dolly's birth immediately raised the question of human cloning. In principle, a human baby could be made using SCNT. The technical obstacles are, however, greater than most people recognize. Experts in the field doubt that human reproductive cloning can be safely pursued, at least for several decades. In Dolly's case, it took 277 attempts to create one live and apparently healthy sheep, a risk level that is clearly unacceptable for human reproduction. More important, the state of Dolly's health is not fully known. One fear associated with cloning is that the clone, having nuclear DNA that may be many years old, will age prematurely, at least in some respects. Mammalian procreation is a profoundly complicated process, as yet little understood, with subtlety of communication between sperm, egg, and chromosomes, which allows DNA from adults to turn back its clock and become, all over again, the DNA of a newly fertilized egg, an embryo, a fetus, and so forth through a complex developmental process. Using cloning to produce a healthy human baby who will become a healthy adult is decidedly beyond the ability of science as of 2002. Expert panels of scientists all strongly condemn the use of SCNT to produce a human baby.

Therapeutic cloning

Cloning, however, may have other human applications beside reproduction, and many scientists endorse these. Usually such applications are referred to as therapeutic cloning, but it should be noted that much research must occur before any therapy can be achieved. Especially interesting is the possibility of combining nonreproductive cloning with embryonic stem cell technologies. Human embryonic stem cells, first isolated in 1998, appear promising as a source of cells that can be used to help the human body regenerate itself. Based on research performed in mice and rats, scientists are optimistic that stem cells may someday be implanted in human beings to regenerate cells or tissues, perhaps anywhere in the body, possibly to treat many conditions, ranging from diseases such as Parkinson's to tissue damage from heart attack.

Embryonic stem cells are derived from embryos, which are destroyed in the process. Some scientists are hopeful that they will be able to find stem cells in the patient's own body that they can isolate and culture, then return to the body as regenerative therapy. Others think that stem cells from embryos are the most promising for therapy. But if implanted in a patient, embryonic stem cells would probably be rejected by the patient's immune system. One way to avoid such rejection, some believe, is to use SCNT. An embryo would be created for the patient using the patient's own DNA. After a few days, the embryo would be destroyed. The stem cells taken from the embryo would be cultured and put into the patient's body, where they might take up the function of damaged cells and be integrated into the body without immune response.

Religious concerns about cloning

While many believe the potential benefits justify research in therapeutic cloning, some object on religious grounds. Many Roman Catholic and Orthodox Christians reject this whole line of research because it uses embryos as instruments of healing for another's benefit rather than respecting them as human lives in their own right. Others believe that if nonreproductive cloning is permitted, even to treat desperately ill patients, then it will become impossible to prevent reproductive cloning, and so they want to hold the line against all human uses of SCNT. A few Protestant and Jewish groups and scholars have given limited approval to nonreproductive cloning.

Outside the United States, most countries with research in this area reject reproductive cloning but permit cloning for research and therapy. In the United States, federal funding is not available as of 2002 for any research involving human embryos. Privately funded research, however, faces no legal limits, even for reproductive cloning. In 2001, one U.S. corporate laboratory, Advanced Cell Technology, published its work, largely unsuccessful, to create human cloned embryos in order to extract stem cells. Some religious leaders object to this situation in which privately funded research is left unregulated.

When it comes to reproductive cloning, religious voices are nearly all agreed in their opposition, although they may give different reasons. Aside from a few isolated individuals, no one has offered a religious argument in support of reproductive cloning. All religious voices agree with the majority of scientists in their objection to cloning based on the medical risk that it might pose for the cloned person, who, even if born healthy, may experience developmental problems, including neurological difficulties, later in life. Until it is known that these risks are not significantly higher for the clone than for someone otherwise conceived, most scientists and ethicists agree that researchers have no right to attempt cloning.

Some religious scholars and organizations oppose cloning as incompatible with social justice. As an exotic form of medicine that benefits the rich, cloning should be opposed in favor of more basic health care and universal access to it.

Others oppose reproductive cloning because it goes against the nature of sexual reproduction, which has profound benefits for a species. Human beings are sexual beings, it is argued, and the necessity of sex for procreation is grounded in hundreds of millions of years of evolution and should not be lightly cast aside by technological innovation. Transcending the biological advantage of sexual procreation, some argue, are the moral and spiritual advantages of the unity of male and female in love, from which a new life emerges from the openness of being, far more than from the designs of will.

Some believe that cloning would confuse and probably subvert relationships between parents and their cloned children. If one person in a couple were the source of the clone's DNA, at a genetic level that parent would be a twin of the clone, not a parent. Whether biological confusion would amount to psychological or moral disorder is of course debatable, but any test might result in tragic consequences. Furthermore, cloning creates a child with nuclear DNA that, in some way at least, is already known. This nuclear DNA begins a new life, not with the usual uncertainties of sexual recombination but through the controls of technology. Many have said that the power to create a clone gives parents far too much power to define their children's genetic identity. Unlike standard reproductive medicine, even if combined in the future with technologies of genetic modification, cloning allows parents to specify that their child will have exactly the nuclear DNA found in the clone's original. This is assuredly not to say that parents may thereby select or control their child's personality or abilities, because persons are more than genes. But some fear that by its nature cloning moves too far in the direction of control and away from the unpredictability of ordinary procreation, so far in fact that a normal parent-child relationship cannot emerge in its proper course. To move in that direction at all is to risk subverting the virtues of parenting, such as unqualified acceptance.

Finally, some have held that cloning will place an unacceptable burden on the cloned child to fulfill the expectations that motivated their cloning in the first place. The fact that the parents may have some prior knowledge of how the clone's nuclear DNA was lived by the clone's original will lead the clone to think that the parents want a child with just these traits. One can imagine that clones will believe they are accepted and loved because they fulfill expectations and not because of their own unique and surprising identity.

In time, reproductive cloning may be widely accepted, much as in vitro fertilization has become accepted. But within religious communities, opposition to cloning is so strong that it is hard to imagine that religious people will ever accept it as a morally appropriate means of human procreation. Nevertheless, despite the strength of the objections, many recognize that human reproductive cloning will occur in time, and when it does the religious concern will shift from preventing cloning to affirming the full human dignity of the clone.

See also Animal Rights; Biotechnology; DNA; Genetic Engineering; Reproductive Technology; Stem Cell Research

Bibliography

brannigan, michael c., ed. ethical issues in human cloning: cross-disciplinary perspectives. new york: seven bridges press, 2001.

bruce, donald, and bruce, ann, eds. engineering genesis: the ethics of genetic engineering in non-human species. london: earthscan, 1998.

cole-turner, ronald, ed. human cloning: religious responses. louisville, ky.: westminster john knox press, 1997.

cole-turner, ronald, ed. beyond cloning: religion and the remaking of humanity. harrisburg, pa.: trinity press international, 2001.

hanson, mark j., ed. claiming power over life: religion and biotechnology policy. washington, d.c.: georgetown university press, 2001.

kass, leon r., and wilson, james q. the ethics of human cloning. washington, d.c.: aei press, 1998.

mcgee, glenn, ed. the human cloning debate. berkeley, calif.: berkeley hills books, 2000.

nussbaum, m. c., and sunstein, c. r., eds. clones and clones: facts and fantasies about human cloning. new york: norton, 1998.

pence, gregory e. who's afraid of human cloning? lanham, md.: rowman and littlefield, 1998.

pence, gregory e., ed. flesh of my flesh: the ethics of cloning humans. lanham, md.: rowman and littlefield, 1998.

ruse, michael, and sheppard, aryne, eds. cloning: responsible science or technomadness? amherst, n.y.: prometheus, 2001.

ronald cole-turner

Cloning: Ethical Issues

Cloning is the creation of an individual that is a genetic replica of another individual. The process transfers a nucleus from a somatic nonreproductive cell into an "enucleated" fertilized egg, one that has had its own nucleus destroyed or removed. The genes in the transferred nucleus then direct the development of a complete organism from the altered fertilized egg. Two individuals who are clones have identical genes in their cell nuclei, but differ in characteristics that are acquired in other ways.

Cloning in Context

Cloning is a natural phenomenon in species as diverse as armadillos, poplar trees, aphids, and bacteria. Identical twins are clones. Biologists have been cloning some organisms, such as carrots, for decades. Attempts to clone animals have been far less successful. They began long before the February 1997 announcement of the birth of Dolly, a sheep cloned from a mammary gland cell nucleus of a six-year-old sheep.

Oxford University developmental biologist John Gurdon cloned frogs in the 1960s, but in a limited way. He showed that a nucleus from a tadpole's intestinal lining cell could be transferred to an enucleated fertilized egg and support development to adulthood, and that a nucleus from an adult cell could support development as far as the tadpole stage. However, he was unable to coax a nucleus from an adult amphibian's cell to support development all the way to adulthood. In the 1980s several companies tried to commercialize cloning of livestock from nuclei taken from embryos or fetuses. The efforts failed because the cloned animals were nearly always very unhealthy newborns and did not survive for long. Currently, livestock cloning is limited to research, although some companies offer tissue preservation services in anticipation of future advances in commercial livestock cloning. There is no reason to believe that human clones would fare any better in terms of health or survivability than most cloned animals do.

The Cloning Ban

Ethical concerns about whether an action is "right" or "wrong" are often clouded by subjectivity, emotion, and perspective. Cloning members of an endangered species, for example, is generally regarded as a positive application of the technology, whereas attempting to clone an extinct woolly mammoth from preserved tissue elicits more negative responses, including that this interferes with nature. A project at Texas A&M University, funded by a dog lover wishing to clone a beloved deceased pet, announced the first successful cloning of a domestic animal, a cat, in February 2002. Cloning pets when strays crowd shelters might be seen as unethical. A different set of ethical issues emerges when considering the cloning of humans, which a few scientists and physicians have proposed doing outside of the United States.

Bioethics is concerned with the rights of individuals, such as the right to privacy and the right to make informed medical decisions. It is difficult to see how these issues would apply to cloning, unless someone was forced or paid to provide material for the procedure, or if an individual was cloned and not informed of his or her origin. Ethical objections to cloning seem to focus more on the fact that this is not a normal way to have a baby. Accordingly, the U.S. House of Representatives voted overwhelmingly on July 31, 2001 to pass legislation that would outlaw human cloning for any reason. However, the broadness of this action may impede other types of medical research, thus introducing a different bioethical dilemma.

The legislation seeks to ban all human cloning, both "reproductive cloning" that would be used to create a baby, and "therapeutic cloning." In therapeutic cloning, a nucleus from a somatic cell is transferred to an enucleated donor egg, and an embryo is allowed to develop for a few days. Then, cells from a part of the embryo called the inner cell mass are used to establish cultures of embryonic stem cells that are genetically identical to the individual who donated the somatic cell nucleus.

If this person has a spinal cord injury or a neurodegenerative disease, the embryonic stem cells might specialize into needed neural tissue. To treat muscular dystrophy, the cells might be coaxed to differentiate into muscle-cell precursors. Such tailored embryonic stem cells would have many applications, and a person's immune system would not reject what is essentially its own tissue. Some people argue that therapeutic cloning violates the rights of early-stage embryos; others argue that banning this research violates the rights of people who might benefit from embryonic stem cell therapy.

According to the bill's ban on producing or selling "any embryo produced by human cloning," scientists caught in the act could expect a fine of up to $1 million or ten years in prison. Proposals to exempt therapeutic cloning were defeated. The criminalization of basic research is unprecedented: Before 2001, bans on using embryonic stem cells applied only to federally funded research, and work using a small number of previously existing stem cell lines was permitted. Since the 2001 ruling, some researchers have moved to nations that permit them to derive new embryonic stem cell lines. Stem cells that are normal parts of adult bodies are being investigated as alternative sources of replacement tissues.

Cloning Misconceptions

The premise that a clone is an exact duplicate of another individual is flawed, and so if the intent of cloning is to create such a copy, it simply will not work. For example, the tips of chromosomes, called telomeres , shorten with each cell division. A clone's telomeres are as short as those from the donor nucleus, which means that they are "older" even at the start of the clone's existence. DNA in the donor nucleus has also had time to mutate, that is to say, it has had time to undergo modification from its original sequence, thus distinguishing it genetically from other cells of the donor. A mutation that would have a negligible or delayed effect in one cell of a many-celled organism, such as a cancer-causing mutation, might be devastating if an entire organism develops under the direction of that nucleus. Finally, the clone's mitochondria , the cell organelles that house the reactions of metabolism and contain some genes, are those of the recipient cell, not the donor, because they reside in the cytoplasm of the egg. Mitochondrial genes, therefore, are different in the clone than they are in the nucleus donor. The consequences of nuclear and mitochondrial genes from different individuals present in the same cell are not known, but there may be incompatibilities.

Perhaps the most compelling reason why a clone is not really a duplicate is that the environment affects gene expression. Cloned calves have different color patterns, because when the animals were embryos, the cells that were destined to produce pigment moved in different ways in each calf. For humans, consider identical twins. Nutrition, stress, exposure to infectious diseases, and other environmental factors greatly influence our characteristics. For these reasons, cloning a deceased child, the application that most would-be cloners give for pursuing the technology, would likely lead to disappointment.

Bioethical concerns over cloning may be moot, because the procedure is extremely difficult to do. Dolly was one of 277 attempts; Cumulina, the first cloned mouse, was among 15 liveborn mice from 942 tries. Cloning so often fails, researchers think, because it is not a natural way to start the development of an animal. That is, the DNA in a somatic cell nucleus is not in the same state as the DNA in a fertilized ovum . The donor DNA in cloning does not pass through an organism's germ line, the normal developmental route to sperm or egg, where gene activities are regulated as a new organism develops.

Ethical objections to human cloning are more philosophical than they are practical. The very idea of cloning assumes that our individuality can be understood so well that we can duplicate it. If human cloning ever became a reality, that this is not true would become evident. After all, we are more than a mere collection of genes.

see also Biotechnology: Ethical Issues; Cloning Genes; Cloning Organisms; Mitochondrial Genome; Stem Cells; Telomere.

Ricki Lewis

Bibliography

Annas, George J. "Cloning and the U.S. Congress." The New England Journal of Medicine 346 (2002): 1599.

Holden, Constance. "Would Cloning Ban Affect Stem Cells?" Science 293 (2001): 1025.

Lewis, Ricki. "The Roots of Cloning." In Discovery: Windows on the Life Sciences. Medford, MA: Blackwell Science, 2000.

Mayor, Susan. "Ban on Human Reproductive Cloning Demanded." British Medical Journal 322 (Jun., 2001): 1566.

Cloning: Applications to biological problems

Human proteins are often used in the medical treatment of various human diseases. The most common way to produce proteins is through human cell culture , an expensive approach that rarely results in adequate quantities of the desired protein. Larger amounts of protein can be produced using bacteria or yeast . However, proteins produced in this way lack important post-translational modification steps necessary for protein maturation and proper functioning. Additionally, there are difficulties associated with the purification processes of proteins derived from bacteria and yeast. Scientists can obtain proteins purified from blood but there is always risk of contamination . For these reasons, new ways of obtaining low-cost, high-yield, purified proteins are in demand.

One solution is to use transgenic animals that are genetically engineered to express human proteins. Gene targeting using nuclear transfer is a process that involves removing nuclei from cultured adult cells engineered to have human genes and inserting the nuclei into egg cells void of its original nucleus .

Transgenic cows, sheep, and goats can produce human proteins in their milk and these proteins undergo the appropriate post-translational modification steps necessary for therapeutic efficacy. The desired protein can be produced up to 40 grams per liter of milk at a relatively low expense. Cattle and other animals are being used experimentally to express specific genes, a process known as "pharming." Using cloned transgenic animals facilitates the large-scale introduction of foreign genes into animals. Transgenic animals are cloned using nuclear gene transfer, which reduces the amount of experimental animals used as well as allows for specification of the sex of the progeny resulting in faster generation of breeding stocks.

Medical benefits from cloned transgenic animals expressing human proteins in their milk are numerous. For example, human serum albumin is a protein used to treat patients suffering from acute burns and over 600 tons are used each year. By removing the gene that expresses bovine serum albumin, cattle clones can be made to express human serum albumin. Another example is found at one biotech company that uses goats to produce human tissue plasminogen activator, a human protein involved in blood clotting cascades. Another biotech company has a flock that produces alpha-1-antitrypsin, a drug currently in clinical trials for the use in treating patients with cystic fibrosis. Cows can also be genetically manipulated using nuclear gene transfer to produce milk that does not have lactose for lactose-intolerant people. There are also certain proteins in milk that cause immunological reactions in certain individuals that can be removed and replaced with other important proteins.

There is currently a significant shortage of organs for patients needing transplants. Long waiting lists lead to prolonged suffering and people often die before they find the necessary matches for transplantation. Transplantation technology in terms of hearts and kidneys is commonplace, but very expensive. Xenotransplantation, or the transplantation of organs from animals into humans, is being investigated, yet graft versus host rejection remains problematic. As an alternative to xenotransplantation, stem cells can be used therapeutically, such as in blood disorders where blood stem cells are used to deliver normal blood cell types. However, the availability of adequate amount of stem cells is a limiting factor for stem cell therapy.

One solution to supersede problems associated with transplantation or stem cell therapy is to use cloning technology along with factors that induce differentiation. The process is termed, "therapeutic cloning" and might be used routinely in the near future. It entails obtaining adult cells, reprogramming them to become stem cell-like using nuclear transfer, and inducing them to proliferate but not to differentiate. Then factors that induce these proliferated cells to differentiate will be used to produce specialized cell types. These now differentiated cell types or organs can then be transplanted into the same donor that supplied the original cells for nuclear transfer.

Although many applications of cloning technology remain in developmental stages, the therapeutic value has great potential. With technological advancements that allow scientists to broaden the applications of cloning becoming available almost daily, modern medicine stands to make rapid improvements in previously difficult areas.

See also DNA hybridization; Immunogenetics; Microbial genetics; Transplantation genetics and immunology

cloning is the generation of genetically identical organisms: each group of such organisms is a clone. Ever since Aldous Huxley's Brave New World, cloning and clones have been the subject both of science fiction and of serious public concern over their possible biotechnological applications. Before taking a paranoid view, however, it is worth noting that clones occur widely and naturally. Many plant varieties are propagated as clones (for instance by grafting) and the summer aphids preying upon them are asexually produced, genetically identical individuals — clones. Identical twins are clones, and the famous Dionne quintuplets born in Canada in 1934 represent a human clone of five people.

Sexual reproduction involves a re-assortment of the genetic material from the two parents and hence the generation of new, genetically distinct individuals. In contrast to this, methods of asexual reproduction result in the production of genetically identical individuals. Bacteria, yeast, and the individual cells of multicellular organisms are able to reproduce asexually, and the products of such replication are clones. Thus, for instance, all the cells in a multicellular organism represent one clone derived from the fertilized egg. During the process of development, and indeed at later stages of life, there may be stably inherited restrictions on the use of the genetic material or new mutations which define new clonally-related groups of cells.

The cells of malignant tumours, for instance, usually carry numbers of mutations which were not originally present in the normal cells of the individual; as these cancer cells progress newer mutations may arise so that several discernibly different clones of cells may be found. One question of interest would be whether all the cells arise from one single event — is the tumour a clone? This question may be addressed in individuals where there is already more than one distinguishable clone of cells present. In women, one of the two X chromosomes will have been inactivated early in development in a random but stable manner. This results in all the tissues being a mosaic of two alternative types of cell. Tumours typically display a single type, demonstrating their clonal origin from a single precursor cell.

This illustrates another important aspect of cloning: the origin of the clone purifies it from a mixed population. For example, many cultivated plants are deliberately propagated asexually by cuttings or grafting, so that one particular variety may be maintained. In molecular biology, this property — that the isolation of a clone selects, maintains, and propagates as a single pure variant — is used directly for analysis of the genetic material itself; the DNA. Pieces of DNA are inserted into a bacterial or viral host in a form that replicates asexually. One single cell is used to start a colony — a clone — and thus large amounts of a single purified DNA fragment may be isolated.

All the cells of a multicellular organism arising from one fertilized egg are clones and, unless subsequently modified, contain the same genetic information. This was demonstrated in plants by regeneration of a whole plant from a single cell from a carrot root. In animals it was shown possible to transplant the nucleus from a gut cell of a tadpole into a fertilized egg, which had had its own nucleus destroyed, and regenerate a new tadpole which now had the genetics of the donor nucleus. Such cloning was first attempted for mammals using mice, but this did not work with any nuclei other than those from the earliest embryos. In the 1990s, however, Ian Wilmut and a team at the Roslin Research Institute in Edinburgh demonstrated a technique allowing nuclei from cells in tissue culture to be used to clone a sheep. They have now demonstrated that these tissue culture cells can be derived from an adult sheep.

The lamb (named Dolly), who was produced from a nucleus from a cell grown from the breast tissue of an adult sheep, has had major political impact as it is now clear that there is no theoretical reason why this cloning should not be possible not only with sheep but with other mammals, including humans. Cloning people is illegal in Britain, but world-wide legislation is not in place. In some quarters it is argued, however, that the technique per se might be useful to regenerate transplant tissues or organs without ever compromising the ethical, legal, and moral susceptibilities that would arise from deliberately generating whole fetuses or people.

Martin Evans

cloning. There is no ‘official’ Buddhist position on cloning nor is one likely to emerge since there is no central authority qualified to speak for the religion as a whole. Based on traditional teachings, however, the attitude of Buddhists in general towards recent advances in genetic engineering is likely to be one of caution. In particular, there are grounds for serious concern surrounding the technique of nucleus substitution. This is when the nucleus of a fertilized egg cell is extracted and replaced with the nucleus of a cell from another being in order to produce a twin of the mature animal, as in the case of Dolly the sheep in 1997.

Buddhists are unlikely to have the same objections to the technique as Christians or other theistic religions. For Christians, to bring into being a new human or animal life by cloning may be seen as usurping the role of the creator. This is not a problem for Buddhism, because in Buddhism the creation of new life is not seen as a ‘gift from God’. For this reason the technique in itself would not be seen as problematic. Furthermore, although Buddhists understand sexual reproduction to be the overwhelmingly most common means by which humans and animals are reborn, it teaches that life can come into being through one of ‘four wombs’ (catur yoni). The last of these refers to the supernatural phenomenon of ‘spontaneous generation’ by which sages and supernatural beings have the power to materialize a human form. Life can thus legitimately begin in more ways than one.

Although the technique of cloning may be morally neutral in itself, there are concerns surrounding the purposes for which it may be used. These centre on the fact that the nature of the technique leads life to be viewed as a product rather than an end in itself. The clone is produced by technicians in a laboratory, and for most of the purposes envisaged so far is then treated as an expendable resource rather than an individual with its own rights and intrinsic dignity. It is hard to see what purposes—scientific or otherwise—can justify the dehumanization that results when life is created and manipulated for other ends. For example, if the clone is to be used to provide spare organs for the person cloned, it would mean that individual life was being produced to be used as a mere instrument for the benefit of another, and effectively treated as property in the way slaves once were. Such dehumanizing techniques would be repugnant to Buddhism, which teaches that individual beings (both human and animal) are worthy of respect in their own right. Buddhism is more concerned about animals than some other religions, and so is likely to be more cautious about the use of animals in experiments of this kind. It should be remembered that Ian Wilmut, the creator of Dolly, failed 276 times before Dolly was conceived. Naturally, in the case of human beings a failure rate of this kind would be even graver, and when weighed against the benefits to be gained from human cloning identified so far the risks do not appear to be justified. In fact, there appears to be no single compelling reason for cloning human subjects. The benefits identified so far fall into two main groups: as an aid to current IVF techniques, and use for genetic selection or eugenics purposes. The numbers who would benefit from the first are very small, and history has shown the potentially grave consequences of the latter. See also Stem Cell Research; Medicine.

Cloning

A clone is a group of genetically identical cells descended from a single common ancestor. Cloning is one method for producing identical twins. After an egg is fertilized, it begins to divide repeatedly. If the egg completely separates during the two-cell stage, identical twins will result. Both individuals will have exactly the same combination of genes (genotype) and each will have the same physical characteristics (phenotype). This is an example of how exact duplicates can naturally occur through sexual reproduction.

Science has capitalized on the mechanisms of cellular reproduction to produce clones. Advances in biotechnology since the 1970s have enabled livestock breeders to clone virtually unlimited numbers of identical animals from a single embryo. This allows the precise duplication of an animal with desired characteristics.

In 1979 veterinarian Steen Willadsen developed a way to divide sheep embryos in half at the two-cell stage, making clones possible. In the next few years, several scientists made further strides in this area with both sheep and cattle embryos. A team developed a simplified method of dividing and cloning sheep embryos in 1984.

Cloning is one area of genetics that is advancing very rapidly, and it is therefore very controversial. If this technology is ever applied to humans, who will decide which genes are "desired" and should be cloned? This is only one of many important questions that have arisen as a result of genetic cloning.

Dairy Farmers Use Cloning Techniques

As an example of cloning techniques, dairy fanners trying to clone a cow with high milk-producing qualities begin by artificially inseminating a high-producing cow with the sperm from a prize bull. The resulting embryo, which contains the entire genetic instructions needed to form a complete calf, develops within its mother. After some time, the embryo divides into a mass of 32 identical cells. The embryo is then carefully removed from the mother cow and separated into 32 separate cells. Finally, after microsurgery on the cells, each new embryo is transplanted into 32 different carrier cows, where it develops fully.

After a normal pregnancy, each carrier cow gives birth to a calf that is genetically identical to the 31 other calves derived from the original 32 cell embryo. Each calf is a clone. The trait for increased milk production has been cloned so that the farmer now has 32 high milk-producing cows instead of just one. Cloning technology has enabled breeders to develop lines of cattle, sheep, and cotton plants that respectively produce more milk, wool, and cotton.

CLONE, CLONING

A clone is a group of organisms that derive from a single ancestor and are genetically identical. A clone can be a group of mammals such as sheep, or a group of cells in culture.

Cloning cells is a powerful tool in biology and medicine, since growing large quantities of identical cells allows for a large harvest of the various identical and useful components of these cells. It is possible to construct genetic components in the laboratory, place them in cells, and then have the cells grow and multiply to produce large quantities of the components.

Cloning is an essential technique in modern molecular biology; it is used widely in studying genetic effects in the drug-abuse field. Cloning much larger organisms such as cows and sheep is expected to have a major impact in that production of the best of any species can theoretically be accomplished by cloning. This is an important goal in agriculture today.

Michael J. Kuhar

cloning vector See vector.

cloning see clone .