ēThree Ways to Clone Mammals

There are basically three ways to clone mammals.

  1. Splitting off a cell from an embryo (twinning)
  2. The Roslin technique used to create Dolly
  3. The Honolulu technique
Let's explain embryo twinning to create clones:
Once an egg has been fertilized by sperm it soon starts dividing.  If it divides into an eight cell embryo and those eight cells are separated, those cells can be implanted into the uteri of eight separate mothers. Then eight clones will be born of different mothers.

Let's explain nuclear transfer, one of the steps in creating Dolly:
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. A technique known as twinning exists, but can only be used before an organismís cells differentiate. All cloning experiments of adult mammals have used a variation of nuclear transfer.
Nuclear transfer requires two cells, a donor cell and an oocyte, or egg cell. 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.

The nucleus is removed from the egg cell >>>>

This eliminates the majority of its genetic information. The donor cell is then forced into the Gap Zero, or G0 cell stage, a dormant phase, in different ways depending on the technique. This dormant phase 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 cellís nucleus is then placed inside the egg cell, either through cell fusion or transplantion. The egg cell is then prompted to begin forming an embryo. When this happens, the embryo is then transplanted into a surrogate mother. If all is done correctly, occasionally a perfect replica of the donor animal will be born.

Each group of researchers has its own specific technique. The best known is the Roslin technique, and the most effective and most recently developed is the Honolulu technique.

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.

The donor cell is grown in a petri/culture dish.

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 culture dish barely has 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.

The enucleated egg cell and the mammary cell are fusing together.

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.

Let's explain the Honolulu technique:
In July of 1998, a team of scientists at the University of Hawaii announced that they had produced three generations of genetically identical cloned mice.
The technique is accredited to Teruhiko Wakayama and Ryuzo Yanagimachi of the University of Hawaii. Mice had long been held to be one of the most difficult mammals to clone due to the fact that almost immediately after a mouse egg is fertilized, it begins dividing. Sheep were used in the Roslin technique because their eggs wait several hours before dividing, possibly giving the egg time to reprogram its new nucleus. Even without this luxury, Wakayama and Yanagimachi were able to clone with a much higher success rate (three clones out of every one-hundred attempts) than Ian Wilmut (one in 277).

Wakayama approached the problem of synchronizing cell cycles differently than Wilmut. Wilmut used udder cells, which had to be forced into the G0 stage. Wakayama initially used three types of cells, Sertoli cells, brain cells, and cumulus cells. Sertoli and brain cells both remain in the G0 state naturally and cumulus cells are almost always in either the G0 or G1 state.

Unfertilized mouse egg cells were used as the recipients of the donor nuclei. After being enucleated, the egg cells had donor nuclei inserted into them. The donor nuclei were taken from cells within minutes of the cellís extraction from a mouse. Unlike the process used to create Dolly, no in vitro, or outside of an animal, culturing was done on the cells. After one hour, the cells had accepted the new nucleus. After an additional five hours, the egg cell was then placed in a chemical culture to jumpstart the cellís growth, just as fertilization does in nature.

In the culture was a substance (cytochalasin B) which stopped the formation of a polar body, a second cell which normally forms before fertilization. The polar body would take half of the genes of the cell, preparing the other cell to receive genes from sperm.

After being jumpstarted, the cells develop into embryos. These embryos can then be transplanted into surrogate mothers and carried to term. The most successful of the cells for the process were cumulus cells, so research was concentrated on cells of that type.

After proving that the technique was viable, Wakayama also made clones of clones and allowed the original clones to give birth normally to prove that they had full reproductive functions. At the time he released his results, Wakayama had created fifty clones.

This new technique allows for further research into exactly how an egg reprograms a nucleus, since the cell functions and genomes of mice are some of the best understood. Mice also reproduce within months, much more rapidly than sheep. This aids in researching long term results.

This web page is courtesy of the people at Kayotic Development. The graphics and text above belong to them.  Therefore, copyright 1998 by team 24355 and Kayotic Development. Their website is http://library.advanced.org/24355/homenav.html.  You can thank them via e-mail at 24355@advanced.org.  Please reference this page as:
Kayotic Development: "The Three Ways to Clone Mammals." Published by the Human Cloning Foundation 03/03/1999, available at http://www.humancloning.org/threeway.htm.