Image: PROBIO AMERICAN, INC

THREE BROWN MICE. The two brown agouti mice on the bottom are clones of the agouti on top. This adult donated a nucleus that was injected into an egg taken from the black mouse next to it. The resulting embryo was transferred to the albino, who acted as a surrogate mother.

This time, the creators of Dolly the cloned sheep, have truly been outdone. An international team of scientists led by Ryuzo Yanagimachi of the University of Hawaii have now created multiple clones and clones of those clones. A paper describing their success appeared in the journal Nature on July 23.

The group is the first to duplicate mammals in a reproducible fashion. Although several researchers have formed single clones from adult animals in recent years--most notably, the sheep dubbed Dolly and Polly, and Gene, a calf--none produced many new animals at once. Yanagimachi and colleagues, however, made more than 50 identical mice that genetically match their sister/parent, sister/grandparent and sister/great grandparent.

The mice were all cloned by way of a new method, called the Honolulu technique--developed by Teruhiko Wakayama, a postdoctoral scientist in Yanagimachi's lab. Whereas earlier clones resulted either from injecting or fusing embryonic cells or from fusing adult cells, the Hawaii mice were cloned by injecting the nucleus from an adult cell into an egg cell, or oocyte, from which the nucleus had already been removed.

Using a special pipette, the researchers injected donor nuclei--taken from cumulus cells that surround developing eggs in ovaries--into oocytes. Next they prevented the eggs from dividing and forming multicelled blastocysts for anywhere from one to six hours.

This delay was a crucial step. "We discovered that a relatively high proportion of the oocytes developed into blastocysts and then further developed when we included a delay," Yanagimachi explained. Learning the molecular events of this delay should shed light on the cellular changes needed to prompt development.

Image: PROBIO AMERICAN, INC DONOR NUCLEUS. The researchers injected nuclei taken from the adult cumulus cells in certain mice into oocytes taken from other mice to create blastocysts, which were then transferred to yet another group of animals to carry them to term.

In the first round of experimentation, the investigators transferred 142 blastocysts into 16 surrogate mice. Some 8.5 to 11.5 days later, five live and five dead fetuses remained. Next, they scaled the test up to include 800 blastocysts and 54 surrogates, yielding 17 live fetuses--10 of which survived. The first born, named Cumulina, and her nest mates all produced normal, fertile offspring.

To prove that they were indeed creating genetic doubles, Yanagimachi's team then turned to different breeds. They took donor nuclei from agouti mice, which are brown, and injected them into black mice. The resulting blastocysts--298 in all--were transferred to 18 albino foster mothers, who birthed five live agouti mice. DNA typing demonstrated that the genetic makeup of the offspring indeed matched that of the agouti donors. Clones of these clones were also genetically identical.

Yanagimachi estimates that for every 100 blastocysts transferred by way of the Honolulu technique to surrogate wombs, 71 take, 5 to 16 fetuses form and 2 to 3 live mice are born. Such efficiency is good news for academic and pharmaceutical researchers alike. Clans of cloned animals should help scientists uncover the cellular and molecular activities involved in development, aging and various diseases.

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