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SOMATIC CELL NUCLEAR TRANSFER
HONOLULU METHOD OF CLONING
Somatic Cell Nuclear Transfer (SCNT) Method
First explored by Hans Spemann in the 1920's to conduct genetics research, nuclear transfer is the technique currently used in the cloning of adult animals. All cloning experiments of adult mammals have used a variation of nuclear transfer.
A somatic cell is any cell other than a sperm, egg, or cell that gives rise to a sperm or egg. Nuclear transfer requires two cells, a donor cell and an oocyte, or egg cell. The nucleus of the egg (containing its DNA) is removed and replaced with the nucleus (and its DNA) of a somatic cell (such as skin or blood) from the recipient. Research has proven that the egg cell works best if it is unfertilized, because it is more likely to accept the donor nucleus as its own. The egg cell must be enucleated, which eliminates the majority of its genetic information. The donor cell is then forced into the Gap Zero, or G0 cell stage, a dormant phase, which causes the cell to shut down but not die. In this state, the nucleus is ready to be accepted by the egg cell. The donor cell's nucleus is then placed inside the egg cell, either through cell fusion or transplantation. The egg cell is then prompted to begin forming an embryo. (To harvest stem cells, the egg containing the transferred nucleus is encouraged to divide until it reached the blastocyst stage, at which time the cells of the inner cell mass are removed and cultured. These are known as embryonic stem cells, or ESC's.) The embryo is transplanted into a surrogate mother if stem cells are not the goal. If all is done correctly, occasionally a perfect replica of the donor animal will be born.
Adult and Fetal Stem Cells Embryonic Stem Cells Embryonic Stem Cells Produced with the SCNT Technique Reproductive Cloning: Embryos produced with SCNT Technique
Purpose of use To obtain undifferentiated stem cells for research and therapy To obtain undifferentiated stem cells for research and therapy To obtain undifferentiated stem cells that are genetically matched to recipient for research and therapy To produce embryo for implantation, leading to birth of a child
Starting material Isolated stem cells from adult or fetal tissue Cells from an embryo at blastocyst stage produced by fertilization Cells from a blastocyst produced by development of an enucleated egg supplied from patient's somatic cell Enucleated egg supplied with nucleus from donor's somatic cell (SCNT technique)
End Product Cells produced in culture to replenish diseased or injured tissue Cells produced in culture to replenish diseased or injured tissue Cells produced in culture to replenish diseased or injured tissue Embryo derived from development of egg
Source: Stem Cells and the Future of Regenerative Medicine, Committee on the Biological and Biomedical Applications of Stem Cell Research, Board on Life Sciences National Research Council Board on Neuroscience and Behavioral Health Institute of Medicine, National Academy Press, Washington, D.C., 2001
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The cloning of Dolly has been the most important event in cloning history. Not only did it spark public interest in the subject, but it also proved that the cloning of adult animals could be accomplished. Previously, it was not known if an adult nucleus was still able to produce a completely new animal. Genetic damage and the simple deactivation of genes in cells were both considered possibly irreversible.
The realization that this was not the case came after the discovery by Ian Wilmut and Keith Cambell of a method with which to synchronize the cell cycles of the donor cell and the egg cell. Without synchronized cell cycles, the nucleus would not be in the correct state for the embryo to accept it. Somehow the donor cell had to be forced into the Gap Zero, or G0 cell stage, or the dormant cell stage.
First, a cell (the donor cell) was selected from the udder cells of a Finn Dorset sheep to provide the genetic information for the clone. For this experiment, the researchers allowed the cell to divide and form a culture in vitro, or outside of an animal. This produced multiple copies of the same nucleus. This step only becomes useful when the DNA is altered, such as in the case of Polly, because then the changes can be studied to make sure that they have taken effect.
A donor cell was taken from the culture and then starved in a mixture which had only enough nutrients to keep the cell alive. This caused the cell to begin shutting down all active genes and enter the G0 stage. The egg cell of a Blackface ewe was then enucleated and placed next to the donor cell. One to eight hours after the removal of the egg cell, an electric pulse was used to fuse the two cells together and, at the same time, activate the development of an embryo. This technique for mimicking the activation provided by sperm is not completely correct, since only a few electrically activated cells survive long enough to produce an embryo.
If the embryo survives, it is allowed to grow for about six days, incubating in a sheep's oviduct. It has been found that cells placed in oviducts early in their development are much more likely to survive than those incubated in the lab. Finally, the embryo is placed into the uterus of a surrogate mother ewe. That ewe then carries the clone until it is ready to give birth. Assuming nothing goes wrong, an exact copy of the donor animal is born.
This newborn sheep has all of the same characteristics of a normal newborn sheep. It has yet to be seen if any adverse effects, such as a higher risk of cancer or other genetic diseases that occur with the gradual damage to DNA over time, are present in Dolly or other animals cloned with this method.
Honolulu Cloning Technique
Full Term Development of Animals from Enucleated Oocytes
Reconstituted with Adult Somatic Cell Nuclei
This technological invention is a proven method of producing live, healthy, cloned male or female offspring from the fibroblast cells of adult animals. The resultant viable offspring are true clones of the adult animal that provided the somatic cells used.
This technology comprises two major steps:
Inserting the nucleus of the somatic cell into the cytoplasm of an enucleated oocyte without activating the oocyte to continue development.
Facilitating embryonic development of the reconstituted oocyte to produce a live offspring.
When the donor nucleus has 2n chromosomes, an additional step can be used to suppress the formation of a polar body and maintain the 2n chromosome number. In this case, the microtubule and/or microfilament assembly is disrupted for a period of time.
This animal cloning technique offers the following features:
Donor cells from a post-natal animal may be obtained either from an in vivo source or from a cultured cell line.
Microinjection using a piezo electric micromanipulator enables a donor nucleus to be harvested and injected using a single needle. This is a major improvement over the cell fusion method.
Because the removal and subsequent injection of a donor nucleus are performed as separate steps, this technique allows the harvested nucleic material to be modified prior to microinjection or mixed with reagents before, during or after combination with the enucleated oocyte.
These methods are applicable to the cloning of all animals, including amphibians, fish, birds and mammals.
HONOLULU METHOD OF CLONING