The Honolulu Technique.
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 each cell’s
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 cell’s 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.
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