For example, isogenic groups were reported in murine twins and triplets Kono et al. Similarly, multiple calves ensued from one donor Bondioli et al. Finally, serial cloning of goat nuclei from the cell stage resulted in 45 kids Zang and Li Three identical transgenic goats were cloned from fetal cells bearing the human gene for antithrombin III, and one goat is producing the protein in her milk Baguisi et al. Although cloning of preimplantation mammalian nuclei has been relatively successful, the cloning efficiency from more advanced donor stages, as in amphibians, decreases.
Nevertheless, a cloning efficiency adequate for commercial use has been achieved with fetal lamb and calf fibroblast and muscle cells, as well as with adult sheep mammary gland, mouse cumulus, and calf cumulus and oviductal cells. However, for those persons speculating on the application of cloning to humans, we emphasize the high mortality rate occurring throughout the experimental procedure.
The Dolly experiment, as we will discuss subsequently, began with attempts to fuse a mammary gland cell to an oocyte; the development of this one ewe represents a success rate of only 0. Several studies in sheep and cattle deserve further discussion. Sims and First initially cloned four calves from cultured cells of the inner cell mass of blastocysts. This result established the feasibility of cloning from cultured cells, and it suggested that transgenic clones might be produced by transfecting cultured cells with a foreign gene and then using these cells as donor cells for nuclear transfer.
Indeed, two laboratories have now produced transgenic cloned animals. Schnieke et al. The cultures were then exposed to G, which kills all cells except those expressing neomycin resistance. Only those cells that had integrated the genes for neomycin resistance and, therefore, clotting factor IX survived and were used for nuclear transfer.
After appropriate clinical trials, the protein harvested from the milk will be used to treat hemophiliacs Wilmut In the future, transgenic clones can be designed to produce a variety of complex human proteins for human use.
It should be noted that the production of transgenic animals by cloning is considerably more efficient than by gene injection into the pronuclei of fertilized eggs. There are several reasons for this increased efficiency, including the fact that transgenic cells can be selected in culture before nuclear transfer. Cloning experiments have provided valuable insight into a number of important cellular processes, such as nuclear reprogramming, cellular aging, and genomic imprinting.
The transplanted nuclei resume RNA synthesis at later embryonic stages, at the same time as embryonic nuclei from fertilized eggs begin to synthesize RNAs. This reversibility of nuclear function also applies to specific genes reviewed in Gurdon Moreover, during the first cell cycle of frog nuclear transplants, non-histone proteins move bidirectionally between the transplanted nucleus and the egg cytoplasm, whereas histone proteins primarily move from the cytoplasm into the nucleus Di Berardino and Hoffner , Hoffner and Di Berardino This result suggests that the chromatin proteins are being modified.
Today, techniques are available to analyze the remodeling of chromatin proteins directly. When transcriptionally inactive sperm nuclei were incubated in extracts from activated amphibian eggs, sperm-specific histone proteins were replaced by somatic histones H2A and H2B via the molecular chaperon nucleoplasmin Katagiri and Ohsumi With respect to mammalian nuclei, recent studies in nuclear transplant embryos of mice, rabbit, pig, and cattle confirmed that changes similar to those observed in amphibian nuclear transplants also occur during nuclear reprogramming Fulka et al.
The importance of nuclear reprogramming is emphasized by the fact that incomplete nuclear reprogramming or its failure in amphibian and mammalian nuclear transplants causes abnormal and arrested development reviewed in Di Berardino a. Scientists are only beginning to understand the molecular changes involved in nuclear reprogramming, yet this line of basic research may result in some of the most beneficial applications of cloning to humans.
For example, if scientists could explain in molecular terms how a differentiated nucleus is de-differentiated, it might be possible to repair certain diseased tissues—a small amount of normal tissue could be removed from a patient and de-differentiated in culture.
After the cell population is expanded, appropriate inducers could be added to promote a desired type of cell differentiation e.
Then, the tissue could be grafted to the patient's diseased areas, where the cells would be recognized as self and not rejected.
Cloning experiments have examined the replication potential of a genome during cellular aging. Normal cells cultured in vitro have a finite replication limit Hayflick and Moorhead However, even after serial cloning through cell cycles, nuclei of blastula cells were still able to direct tadpole development Robert G. McKinnell, unpublished study. After serial transplantation to oocytes, even terminally differentiated erythrocyte nuclei that had gone through more than cell cycles had the competence to direct the formation of tadpoles Hoffner Orr et al.
Further studies may reveal the mechanism of cellular rejuvenation. Telomeres, which are normally reduced in length during the aging process Greider and Blackburn , Shay , may also be shortened in animal clones. Dolly and two other cloned sheep were reported to have telomeres that were shorter than those of age-matched controls Shiels et al.
Despite their shortened telomeres, the cloned sheep were vigorous and healthy. It remains to be seen if the reduced telomere lengths will have an effect during the lifetime of the sheep. Nuclear transfer and molecular studies in mice elucidated genomic imprinting, a genetic mechanism that controls the differential expression of certain pairs of autosomal alleles.
Mouse nuclear transplants constructed of two maternal or two paternal pronuclei fail to develop; only nuclear transplants comprising biparental nuclei form normal offspring McGrath and Softer , Surani et al.
Molecular analyses of mouse embryos revealed that certain autosomal genes are differentially expressed from maternal and paternal genomes at specific times in development Latham Thus, an embryo with two inactive maternal or paternal genes fails to transcribe a necessary gene product. It is for this reason that the mammalian embryo requires a set of genes from both the father and the mother for normal development.
The experimental results in mice led to the clarification of the basis of several inherited human disorders, such as Prader Willi and Angelman syndromes, which present different phenotypes but are both due to deletions in different homologues of chromosome paternal deletions in Prader Willi patients and maternal deletions in Angelman patients Driscoll Because of their fundamental nature, scientific discoveries in the basic sciences e.
Knowledge in itself is amoral, but the choices for its applications reside in the ethical decisions of humans. Cloning, like other developments in basic science, was initiated to seek new fundamental knowledge. In addition to yielding information about the role of the nucleus during cell differentiation, the procedure also provided insight into basic aspects of modifying the cancer phenotype, rejuvenating aged nuclei by oocyte cytoplasm, nuclear reprogramming, and genomic imprinting. It will continue to yield new knowledge in these and other basic subjects.
As scientists who have worked in frog cloning, we are gratified to see decades of basic research culminating in the production of cloned mammals that will produce human proteins for the alleviation of human disease. Cloning will likely result in the genetic improvement of livestock, the production of animal models to study and treat human diseases, and sources of animal tissues and organs for xenotransplantation to humans. We consider these applications of cloning appropriate.
Indeed, they are the reasons why Dolly was produced. Once Dolly appeared, however, the news media became consumed with the idea of cloning human adults, which immediately stimulated a worldwide debate among ethicists, theologians, clerics, lawyers, legislators, and, of course, laypeople. We do not intend to summarize the many proposals and convictions of others; rather, we delineate briefly the reasons for our belief that human cloning is scientifically and ethically unsound.
We define human cloning as the attempt to produce a human organism by any cloning procedure: blastomere isolation, bisection splitting of preimplantation embryos, and nuclear transplantation. Nuclear transplantation of embryonic, fetal, or adult cells from all species results in abnormal animals at a frequency that increases with the age of the donor cell.
The failures result from incomplete nuclear reprogramming and failed cell cycle matching, although other causes may in some cases be responsible. Abnormal nuclear transplants from all donor stages arrest development at various stages: nuclear transfer and activation, cleavage, organo-genesis, tadpole, and juvenile. In viviparous species, abnormal transplant embryos may fail to implant, and those that do implant may abort at various embryonic and fetal stages.
Finally, those nuclear transplants that are born may die soon after birth or survive with birth defects. Furthermore, even some normal nuclear transplant frog blastulae derived from embryonic or adult nuclei contained abnormal chromosomes in some of their cells, whereas the chromosomes in other cells of the same blastulae appeared normal. Such cases would pose confusion in monitoring normal-appearing nuclear transplants for further culture and development.
For these reasons, we consider the use of any cell type not just those from adults for human cloning scientifically and ethically unsound. In the case of Dolly, she was the only successful case out of attempted fusions of oocytes and donor cells that were taken from cultures of mammary gland. Even as cloning of adult nuclei becomes much more efficient, there will still be hazards to humans.
Mutations could also arise in the donor cells during cell culture, an event that is not unusual. There are still other scientific concerns. Will telomere shortening in the donor cell limit the life span of the clone?
Will stored gene products RNAs and proteins in oocytes from foreign donors always be compatible with the donor nucleus? Finally, it is important to consider that meiosis, which precedes sexual reproduction, affords humans another opportunity for DNA repair and therefore should not be avoided in favor of asexual somatic reproduction. For all of these reasons, we oppose human cloning.
Debra L. Mark R. Krampf, Department of Biochemistry, University of Minnesota, provided valuable assistance in preparing several of the illustrations for publication. Because of bibliographic restrictions, the primary references of some authors were not cited, and we apologize for these omissions. Aimar C. Annales d'Embryologie et de Morphogenese 5 : 5 — Google Scholar. Baguisi A et al. Production of goats by somatic nuclear transfer.
Nature Biotechnology 17 : — Production of identical bovine offspring by nuclear transfer. Theriogeneology 33 : — Briggs R King TJ. Nuclear transplantation studies on the early gastrula Rana pipiens. Developmental Biology 2 : — Journal of Experimental Zoology : — Brun R Kobel HR. Revue Suisse de Zoologie 79 : — Campbell KHS.
Nuclear equivalence, nuclear transfer, and the cell cycle. Cloning 1 : 3 — Nuclear transfer in cattle: Birth of cloned calves and estimation of blastomere totipotency in morulae used as a source of nuclei. Cloned transgenic calves produced from nonquies-cent fetal fibroblasts. Science : — Comandon J de Fonbrune P. Journal of Cancer Research and Clinical Oncology : — Di Berardino MA.
Genomic Potential of Differentiated Cells. New York : Columbia University Press. Genomic potential- Acetabularia to mammals. Journal of Cellular Physiology : — Nucleocytoplasmic exchange of non-histone proteins in amphibian embryos.
Experimental Cell Research 84 : — Two additional mules are cloned that same year as part of a joint project between the University of Idaho and Utah State University. August 6, - Scientists at the Laboratory of Reproductive Technology in Cremona, Italy, say they have created the world's first cloned horse, Prometea, from an adult cell taken from the horse who gave birth to her.
February 12, - South Korean researchers falsely report they have created human embryos through cloning and extracted embryonic stem cells. An investigative panel concludes in that the human stem cell cloning research was faked.
August 3, - South Korean researchers announce they have successfully cloned a dog, an Afghan hound named Snuppy. May - A clone of two-time quarter horse world champion Tailor Fit is born, one of several cloned horses born that year. September - At South Korea's Incheon Airport, seven "super clone" sniffer-dogs are dispatched to detect contraband luggage.
They are all golden Labrador Retrievers that are genetically identical to Chase, who was the top drug detention canine until he retired in April - For the first time, cloning technologies are used to generate stem cells that are genetically matched to adult patients. Scientists put the nucleus of an adult skin cell inside an egg and the reconstructed egg went through the initial stages of embryonic development, according to research published in the journal Cell Stem Cell.
Josh Gurdon, a scientist who was also heavily involved in cloning experiments, was able to prove that cloning organisms could work with adult cells that had already differentiated. This development has allowed researchers to perform experiments broadly without having to restrict themselves to using solely embryonic stem cells.
Image illustrates the nuclear transfer technique, developed by Robert Briggs and Thomas King, in which the nucleus of one cell is replaced by the nucleus of another cell. Possibly the most famous cloned animal is Dolly the Sheep. Dolly was created by scientist Ian Wilmut, using the differentiated nuclear transfer technique developed by Josh Gurdon. Electric currents were used to fuse the transferred nucleus and egg together, and the zygote underwent multiple cell divisions.
A black-faced sheep was used as a surrogate mother to carry the developing blastocyst, and she eventually gave birth to a white-faced sheep, proving that the cloning was successful. Dolly was the first-ever cloned mammal, and she was even featured on the cover of Time magazine. She was specifically created for a project involving the production of mammals with desirable proteins in their milk to aid in human medicinal research.
After the successful cloning of Dolly, scientists rushed to clone other mammals with the hopes that they could garner the same amount of attention that Dolly did. Contrary to expectations, as CopyCat grew older, she did not have the same coat coloration as the cat from which she was cloned.
The researchers concluded that the difference in coloring was due to epigenetics, meaning that the fur patterns were influenced by modifications to specific genes. For example, portions of a DNA sequence may have methyl groups attached to them. This can inactivate genes in that region, resulting in a different phenotype from an organism that has the same DNA sequence, but without methylation.
The effect of DNA methylation. Both the yellow and the brown mouse have the exact same DNA sequence. However, due to different environmental conditions within the womb, the DNA of the brown mouse is methylated in different locations from the DNA of the yellow mouse, which resulted in two mice that are phenotypically different.
Thus, while CopyCat and the cat it was cloned from are genetically identical i. This discovery revealed that clones might not always look exactly the same, which was a common misconception. Snuppy, the first successful dog clone, was created by scientists in South Korea using an ear cell from an adult Afghan hound for nuclear transfer. Never before had a mammal been cloned from an adult somatic cell. What was the big deal? However, while embryonic cells are ready to activate any gene, differentiated adult cells have shut down the genes that they don't need for their specific functions.
When an adult cell nucleus is used as a donor, its genetic information must be reset to an embryonic state. Often the resetting process is incomplete, and the embryos fail to develop. Of attempts, only one produced an embryo that was carried to term in a surrogate mother. This famous lamb, named Dolly, brought cloning into the limelight. Her arrival started conversations about the implications of cloning, bringing controversies over human cloning and stem cell research into the public eye.
Primates are good models for studying human disorders. Cloning identical primates would decrease the genetic variation of research animals, and therefore the number of animals need in research studies. The resulting embryos were then implanted into surrogate mothers. Out of 29 cloned embryos, two monkeys were born. One was a female named Neti, and the other was a male named Ditto.
This experiment was an exciting combination of findings from earlier work. Campbell and Wilmut had already created a clone using the nucleus of a cultured cell.
Factor IX codes for a protein that helps blood clot, and it's used to treat hemophilia, a genetic disorder where blood doesn't form proper clots. To create the transgenic sheep, the scientists performed nuclear transfer using donor DNA from the cultured transgenic cells. The result was Polly, a sheep that produced Factor IX protein in her milk. This experiment showed that sheep could be engineered to make therapeutic and other useful proteins in their milk, highlighting the potential medical and commercial uses for cloning.
After the successes leading up to Dolly and Polly, other scientists wanted to see if similar techniques could be used to clone other mammalian species. Before long, several more animals had been successfully cloned. Among them were transgenic animals, clones made from fetal and adult cells, and a male mouse; all previous clones had been female.
As the list of successfully cloned animals grew, scientists began to explore cloning as a way to create animals belonging to endangered or extinct species.
A challenge to cloning endangered and extinct species is finding closely related animals to serve as egg donors and surrogates. The gaur and mouflon were chosen in part because they are close relatives of domestic cattle and sheep, respectively. In , using goast as egg donors and surrogates, another group of researchers cloned the first extinct animal, a Spanish mountain goat called the bucardo.
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