Cloning Organisms

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.